Recombinant nel-like (mell) protein production

ABSTRACT

The present invention provides a method and system for producing a NELL protein. The method and system comprise a CELL encoding a NELL protein or peptide and a non-insect secretory signal peptide.

CROSS-REFERENCE

This application is a Continuation of pending U.S. patent application Ser. No. 15/462,682 filed on Mar. 17, 2017, which is a division of U.S. patent application Ser. No. 13/769,222, filed Feb. 15, 2013, issued as U.S. Pat. No. 9,598,480 on Mar. 21, 2017, which is a Continuation of U.S. patent application Ser. No. 13/769,222, filed Feb. 15, 2013, which is a Continuation of U.S. patent application Ser. No. 13/121,394, filed Jun. 8, 2011, abandoned, which is a 371 U.S. National Phase of PCT/US2009/059893, filed Oct. 7, 2009. The teaching of these applications is incorporated by reference in its entirety.

GOVERNMENT SUPPORT CLAUSE

This invention was made with Government support under Grant Numbers DE000422 and DE014649, awarded by the National Institutes of Health. The Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention is directed to a system and method of producing NELL peptides.

BACKGROUND OF THE INVENTION

Growth factors are substances, such as peptides, which affect the growth and differentiation of defined populations of cells in vivo or in vitro.

Bone formation occurs during development of long bones (endochondral bone formation) and flat bones (intramembranous bone formation). Further, bone formation occurs during bone remodeling which occurs continuously in adult life in order to preserve the integrity of the skeleton. Finally, bone formation occurs during bone repair, such as when bone wounds occur in a fracture or surgical situation, for example. While separate bone formation mechanisms are thought to be involved in the embryological development of long and flat bones and repair is thought to involve intramembranous bone formation.

Bone formation by either mechanism involves the activity of osteoblasts, which are regulated by growth factors. Osteoblasts are derived from a pool of marrow stromal cells (also known as mesenchymal stem cells; MSC). These cells are present in a variety of tissues and are prevalent in bone marrow stroma. MSC are pluripotent and can differentiate into a variety of cell types including osteoblasts, chondrocytes, fibroblasts, myocytes, and adipocytes. Growth factors are thought to impact osteogenic cell proliferation, differentiation and osteoblast mineralization, each of which impacts bone formation.

Autogenous bone has been used, such to repair bone in patients with craniosynostosis and cleft grafting, for example. Craniosynostosis (CS), the premature closure of cranial sutures, affects 1 in 3,000 infants and therefore is one of the most common human congenital craniofacial deformities. Premature suture closure results in cranial dimorphism, which can need surgical correction. Premature suture closure in human CS can occur by two possibly distinct processes: calvarial overgrowth and bony fusion. Recently, fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor 1 (FGFR1) have been implicated in premature cranial suture fusion via CBFA1-mediated pathways (8). Missense mutation of CBFA1 is linked to cleidocranial dysplasia, manifested as delayed suture closure.

Autologous bone grafting procedures have been performed utilizing autogenous bone, such as from the iliac crest or calvaria. These donor sites are not without associated morbidity including pain, gait disturbance, thigh paresthesia for iliac crest donor sites, and infection, neurologic deficits, and hematomas for calvarial grafts. Further, donor sites can have limited volume and can contribute to increased surgical time and hospital stay.

Alloplastic grafting materials have also been utilized, and growth factors have been tested in animal models. For example, bFGF has shown potential for use in bone regeneration and repair. Another family of osteogenic growth factors have been described as bone morphogenic protein (BMP). Specifically, BMP-2 recombinant protein has been demonstrated to regenerate mandibular continuity defects and cleft palate defects with results equal to or better than autogenous particulate bone and marrow. BMPs and other osteogenic factors have been studied for use in clinical applications. However, the cost of using minimally effective dosages of BMP has been a limiting factor in clinical use.

Spinal fusion is a surgical technique in which one more of the vertebrae of the spine are united together so that motion no longer occurs between them. Indications include: treatment of a fractured (broken) vertebra, correction of deformity, elimination of pain from motion, treatment of instability, and treatment of some cervical disc herniations. The surgery can involve placement of a bone graft between the vertebrae to obtain a solid union between the vertebrae. The procedure also can involve supplemental treatments including the placement of plates, screws, cages, and recently bone morphogenic protein 2 and 7 to assist in stabilizing and healing the bone graft. Autogenous bone grafting has been the clinically preferred method, and yet has about a 30-50% failure rate. Autogenous bone grafting is a separate surgery and also carries significant morbidity.

Cartilage is a type of dense connective tissue. It is composed of chondrocytes which are dispersed in a firm gel-like matrix. Cartilage is avascular (contains no blood vessels) and nutrients are diffused through the matrix. Cartilage is found in the joints, the rib cage, the ear, the nose, in the throat and between intervertebral disks. There are three main types of cartilage: hyaline (e.g., costal cartilages, the cartilages of the nose, trachea, and bronchi, and the articular cartilages of joints), elastic (e.g., external ear, external auditory meatus, part of the Eustachian tube, epiglottis, and in some of the laryngeal cartilages) and fibrocartilage [e.g. meniscus (e.g., wrist triangular fibrocartilage complex, knee meniscus), intervertebral discs, temporomandibular joint disc, the pubic symphysis, and in some tendons and ligaments at their attachment to bones. One of the main purposes of cartilage is to provide a framework upon which bone deposition could begin (i.e., during endochondral ossification). Another important purpose of cartilage is to provide smooth surfaces for the movement of articulating bones. For example, articular cartilage, most notably that which is found in the knee joint, is generally characterized by very low friction, high wear resistance, and poor regenerative qualities. It is responsible for much of the compressive resistance and load bearing qualities of the knee joint and, without it, walking is painful to impossible. Yet another important purpose of cartilage is to provide, firm, yet flexible support (e.g., nasal cartilage, spinal discs, tracheal cartilage, knee meniscus, bronchial cartilage). For instance, cartilage such as the meniscus plays a crucial role in joint stability, lubrication, and force transmission. Under a weight bearing load, the meniscus maintains the balanced position of the femur on the tibia and distributes the compressive forces by increasing the surface contact area, thereby decreasing the average stress two to three times. Additionally, the menisci interact with the joint fluid to produce a coefficient of friction that is five times as slick as ice on ice. In another example, the intervertebral disc has several important functions, including functioning as a spacer, as a shock absorber, and as a motion unit. The gelatinous central portion of the disc is called the nucleus pulposus. It is composed of 80-90% water. The solid portion of the nucleus is Type II collagen and non-aggregated proteoglycans. The outer ligamentous ring around the nucleus pulposus is called the annulus fibrosus, which hydraulically seals the nucleus, and allows intradiscal pressures to rise as the disc is loaded. The annulus has overlapping radial bands, not unlike the plies of a radial tire, and this allows torsional stresses to be distributed through the annulus under normal loading without rupture. The disc functions as a hydraulic cylinder. The annulus interacts with the nucleus. As the nucleus is pressurized, the annular fibers serve a containment function to prevent the nucleus from bulging or herniating.

Cartilage can be damaged by wear, injury or diseases. As we age, the water and protein content of the body's cartilage changes. This change results in weaker, more fragile and thin cartilage. Osteoarthritis is a common condition of cartilage failure that can lead to limited range of motion, bone damage and invariably, pain. Due to a combination of acute stress and chronic fatigue, osteoarthritis directly manifests itself in a wearing away of the articulating surface and, in extreme cases, bone can be exposed in the joint. In another example, loss of the protective stabilizing meniscus leads to increased joint laxity or abnormal motions that lead to joint instability. The excessive motion and narrowed contact area promotes early arthritic changes. At the cellular level, there is initially a loss of cells from the superficial layer of the articular cartilage followed by cartilage splitting, subsequent thinning and erosion occurs, and finally protrusion of the underlying raw bone. The earliest arthritic changes have been noted three weeks after loss of the entire meniscus. In yet another example, because both the discs and the joints that stack the vertebrae (facet joints) are partly composed of cartilage, these areas are subject to wear and tear over time (degenerative changes). As the inner nucleus dehydrates, the disc space narrows, and redundant annular ligaments bulge. With progressive nuclear dehydration, the annular fibers can crack and tear. Loss of normal soft tissue tension may allow the spinal segment to sublux (e.g. partial dislocation of the joint), leading to osteophyte formation (bone spurs), foraminal narrowing, mechanical instability, and pain. If the annular fibers stretch or rupture, allowing the pressurized nuclear material to bulge or herniate and compress neural tissues, pain and weakness may result. This is the condition called a pinched nerve, slipped disc, or herniated disc. Radiculopathy refers to nerve irritation caused by damage to the disc between the vertebrae. Mechanical dysfunction may also cause disc degeneration and pain (e.g. degenerative disc disease). For example, the disc may be damaged as the result of some trauma that overloads the capacity of the disc to withstand increased forces passing through it, and inner or outer portions of the annular fibers may tear. These tom fibers may be the focus for inflammatory response when they are subjected to increased stress, and may cause pain directly, or through the compensatory protective spasm of the deep paraspinal muscles.

NELL peptide has been documented to stimulate bone formation and cartilage formation. Existing methods and systems of producing NELL peptides have limited success with problems. A common problem of existing NELL peptide expression systems (e.g., a mammalian cell such as a CHO cell expression system) is low productivity. Low productivity is generally found to be caused by, for example, an inappropriate construct of the nucleic acid sequence expressing a NELL peptide and/or other additional sequences, e.g., a secretory signal peptide sequence, in a particular expression system, e.g., a CHO cell.

Therefore there is a continuous need for a method and system for producing NELL peptides.

SUMMARY OF THE INVENTION

The present invention relates to a system and method for producing a NELL peptide. The method comprises:

providing a nucleic acid construct including at least a nucleic acid encoding at least a NELL peptide in frame with a nucleic acid encoding a non-insect secretory signal peptide;

transfecting a mammalian cell with the nucleic acid construct; and

culturing the mammalian cell under conditions that permit expression of the NELL peptide.

The mammalian cell can be, e.g., a Chinese hamster ovary cell. The nucleic acid encodes NELL1 or NELL2 or both.

In some embodiments, the method further comprises: collecting NELL peptide secreted from the cell line; and substantially purifying the NELL peptide.

In some embodiments, the method of the above various embodiments can further comprise testing the activity of the NELL peptide to induce bone formation.

The act of purifying can be any step of purifying a protein or peptide. In some embodiments, the act of purifying includes chromatography purification.

In some embodiments, the present invention provides a nucleic acid construct for expressing a NELL peptide in a mammalian cell. The nucleic acid construct comprises at least a nucleic acid encoding at least a NELL peptide in frame with a nucleic acid encoding a secretory signal peptide that is a non-insect secretory signal peptide. The nucleic acid encodes NELL1 or NELL2. In some embodiments, the mammalian cell can be a CHO cell.

In some embodiments, the present invention provides a mammalian cell line for expressing a NELL peptide. The cell line comprises a nucleic acid construct comprising at least a nucleic acid encoding at least a NELL peptide in frame with a nucleic acid encoding a secretory signal peptide that is a non-insect secretory signal peptide. The nucleic acid encodes NELL1 or NELL2. In some embodiments, the mammalian cell can be a CHO cell.

In some embodiments, the present invention provides a polypeptide comprising a NELL peptide and a non-insect secretory signal peptide.

In some embodiments, the mammalian cell can be human embryo kidney cell, such as HEK-293.

The present invention provides a method of purifying a NELL peptide, comprising providing a crude solution containing a NELL peptide, and subjecting the crude solution to membrane chromotography to obtain a purified NELL peptide.

In some embodiments, the membrane chromatography is anion-exchange membrane chromatography or cation-exchange membrane chromatography.

The present invention provides a method of purifying a NELLpeptide, comprising providing a crude solution containing a NELL peptide, and subjecting the crude solution to chromotography process to obtain a purified NELL peptide, wherein the chromatography process comprises a medium comprising a metal ion bearing two or more charges. In some embodiments, the metal ion is calcium ion.

The NELL peptide can be NELL-1 peptide or NELL-2 peptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D illustrate a signal peptide-NELL1-FLAG nucleic acid construct. The underlined amino acid sequence is from melittin signal peptide. The NELL1 peptide is from rat NELL1 peptide.

FIG. 2A is an illustration of a CBB-stained SDS-PAGE gel of UnoQ-eluate containing purified NELL1 peptide.

FIG. 2B shows a Western blot using anti-FLAG antibody depicting NELL1-FLAG expression in reference to a protein ladder.

FIG. 2C is an illustration of a CBB-stained SDS-PAGE gel of UnoQ-eluate containing NELL1-FLAG.

FIG. 2D shows a Western blot using anti-FLAG antibody depicting NELL1-FLAG expression.

FIG. 3A depicts the nucleic acid sequence of the cDNA construct and amino acid sequences of three different signal peptides that were used for the constructs.

FIG. 3B is a Western blot with anti-c-myc antibody detecting secreting NELL1 from transfections with different constructs after immunoprecipitation using anti-c-myc agarose.

FIG. 3C is a Western blot with anti-c-myc or mouse anti-human NELL1 antibodies detecting secreting NELL1 after immunoprecipitation using rabbit anti-human Nell-1 antibody-NFIS activated sepharose.

FIGS. 4A-D show NELL1 (homo sapiens) nucleotide sequence and corresponding amino acid sequence (SEQ ID NOs: 1 and 2).

FIGS. 5A-D show NELL1 (rattus norvegicus) nucleotide sequence and corresponding amino acid sequence (SEQ ID NOs: 3 and 4).

FIGS. 6A-F show NELL1 (Mus musculs) nucleotide sequence and corresponding amino acid sequence (SEQ ID NOs: 5 and 6).

FIGS. 7A-D show NELL2 (homo sapiens) nucleotide sequence and corresponding amino acid sequence (SEQ ID NOs: 7 and 8).

FIGS. 8A-D show NELL2 (rattus norvegicus) nucleotide sequence and corresponding amino acid sequence (SEQ ID NOs: 9 and 10).

FIGS. 9A-D show NELL2 (Mus musculs) nucleotide sequence and corresponding amino acid sequence (SEQ ID NOs: 11 and 12).

FIGS. 10A-D show NELL2 (Gallus gallus) nucleotide sequence and corresponding amino acid sequence (SEQ ID NOs: 13 and 14).

DETAILED DESCRIPTION Definitions

As used herein, the terms “polypeptide”, “peptide” and “protein” can be used interchangeably to refer to a polymer of amino acid residues. The terms can apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

The term “antibody” can include various forms of modified or altered antibodies, such as an intact immunoglobulin, an Fv fragment containing only the light and heavy chain variable regions, an Fv fragment linked by a disulfide bond, a Fab or F(ab′)² fragment containing the variable regions and parts of the constant regions, a single-chain antibody and the like. An antibody can include intact molecules as well as fragments thereof, such as, Fab and F(ab′)², and/or single-chain antibodies (e.g. scFv) which can bind an epitopic determinant. An antibody can be of animal, e.g., mouse or rat, or of human origin, or can be chimeric or humanized. Antibodies can be polyclonal or monoclonal antibodies (mAbs), such as monoclonal antibodies with specificity for a polypeptide encoded by a NELL1 or NELL 2 protein.

The term “capture agent” refers to molecules that specifically bind other molecules to form a binding complex such as, but not limited to, an antibody-antigen complex, a lectin-carbohydrate complex, a nucleic acid-nucleic acid complex or a biotin-avidin complex.

The term “specifically binds” refers to the binding property of a biomolecule (e.g., protein, nucleic acid, antibody, etc.), which is determinative of the presence of such a biomolecule in a heterogeneous population of molecules (e.g., proteins and other biologies). Thus, under designated conditions (e.g., immunoassay conditions in the case of an antibody or stringent hybridization conditions in the case of a nucleic acid), the specified ligand or antibody specifically binds to its particular “target” molecule and will not bind in a significant amount to other molecules present in the sample.

The terms “nucleic acid” or “oligonucleotide” refer to at least two nucleotides covalently linked together. A nucleic acid of the present invention can be single-stranded or double stranded and can contain phosphodiester bonds, although in some cases, nucleic acid analogs can be included that can have alternate backbones, comprising, for example, phosphoramide, phosphorothioate, phosphorodithioate, omethylphophoroamidite linkages, and/or peptide nucleic acid backbones and linkages. Analog nucleic acids can have positive backbones and/or non-ribose backbones. Nucleic acids can also include one or more carbocyclic sugars. Modifications of the ribose-phosphate backbone can be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments, for example.

The term “specific hybridization” refers to the preferential binding, duplexing, or hybridizing of a nucleic acid molecule to a particular nucleotide sequence under stringent conditions, including conditions under which a probe can hybridize preferentially to its target subsequence, and can hybridize to a lesser extent to other sequences.

The terms “NELL1 cDNA” refer to SEQ ID NO: 1, 3 and 5, and “NELL2 cDNA” can refer to SEQ ID NO:7, 9, 11 and 13.

NELL Peptides

NELL1 is a 810 aa (amino acid) peptide, distributed primarily in bone. In adults, NELL1 is expressed at high levels in craniofacial bone, and lower levels in long bone. NELL1 has known roles in osteoblast differentiation, bone formation and bone regeneration. NELL1 has known rules in forming cartilage tissues without forming bone.

NELL 2 is a 816 aa peptide, distributed in neural cells and brain.

The human NELL1 gene includes at least 3 Cbfal response elements in the promoter region. Cbfal specifically binds to these response elements. NELL1 expression can be under the control of these transcription factors expressed endogenously at least in preosteoblasts, osteoblasts and hypertrophic chondrocytes in development and in adulthood. Cleidocranial disostosis is a developmental cranial defect thought to be caused at least in part by Cbfa disruption.

A NELL1 peptide can be encoded by the NELL1 gene or cDNA and includes SEQ ID NO: 2, 4, and 6. The NELL1 peptide can include a NELL1 peptide fragment that retains the ability to induce osteogenic cell differentiation, osteoblast differentiation or bone formation. In some embodiments, the NELL1 peptide can include a NELL 1 peptide fragment that retains the ability to induce cartilage formation without forming bone. A NELL2 peptide can be encoded by the NELL2 gene or cDNA and includes SEQ ID NO: 8, 10, 12 and 14. The NELL2 peptide can include NELL2 peptide fragments that retain similar activity to the full NELL2 peptide sequence.

In some embodiments, the amino acid sequence of the NELL peptide can be selected from the group including, but not limited to human NELL1 (SEQ ID NO:2), rat NELL1 (SEQ ID NO:4), mouse NELL1 (SEQ ID NO:6), or human NELL2 (SEQ ID NO:8), rat NELL2 (SEQ ID NO:10), mouse NELL2 (SEQ ID NO:12), chicken NELL2 (SEQ ID NO:14). The amino acid sequence can also include sequences such as those with substantial similarity, such as sequences having at least about 75% sequence similarity with any portion of the sequences listed above, or contain similar active binding domains as NELL1 peptides.

The term “derivative” as used herein, refers to any chemical or biological compound or material derived from a NELL peptide, structural equivalents thereof or conformational equivalents thereof. For example, such a derivative can include any pro-drug form, PEGylated form, or any other form of a NELL peptide that renders the NELL peptide more stable or more osteophilic or lipophilic. In some embodiments, the derivative can be a NELL peptide attached to poly(ethylene glycol), a poly(amino acid), a hydrocarbyl short chain having C1-C20 carbons, or a biocompatible polymer. In some embodiments, the term “derivative” can include NELL peptide mimetics. As used herein, the term “mimetic” refers to a peptide having at least one non-peptide bond in its backbone. A peptide bond is a chemical bond formed between the carboxylic acid group of an amino acid molecule and the amino group of another amino acid molecule. A NELL peptide mimetic can be any compound that exhibits at least one or more bone forming, bone repairing, cartilage forming, and/or cartilage repairing function of a NELL peptide.

Synthetic methods for making peptide mimetics are well known in the art. The following describes an example of the basic procedure for the synthesis of a peptide, including a peptide mimetics:

Before the peptide synthesis starts, the amine terminus of the amino acid (starting material) can protected with FMOC (9-fluoromethyl carbamate) or other protective groups, and a solid support such as a Merrifield resin (free amines) is used as an initiator. Then, step (1) through step (3) reactions are performed and repeated until the desired peptide is obtained: (1) a free-amine is reacted with carboxyl terminus using carbodiimide chemistry, (2) the amino acid sequence is purified, and (3) the protecting group, e.g., the FMOC protecting group, is removed under mildly acidic conditions to yield a free amine. The peptide can then be cleaved from the resin to yield a free standing peptide or peptide mimetics.

Systems Expressing NELL Peptides

Generally, the method of invention includes providing a nucleic acid sequence encoding a NELL peptide, such as NELL1 or NELL2 peptide, in frame with a nucleic acid sequence encoding a non-insect signal peptide.

In one embodiment, the method can include transfecting an insect cell line with a nucleic acid construct encoding a NELL peptide; and culturing the insect cell line under conditions that permit expression and/or secretion of the NELL peptide. For example, the cell line can be transfected transiently or stably with the nucleic acid construct encoding a NELL peptide.

In one embodiment, the method can include providing a nucleic acid sequence encoding a NELL peptide, such as NELL1 or NELL2 peptide. The nucleic acid sequence can be a cDNA or genomic DNA, encoding at least a functional portion of a NELL peptide. For example, the nucleic acid sequence can be selected from the group including, but not limited to human NELL1 (SEQ ID NO: 1), rat NELL1 (SEQ ID NO:3), mouse NELL1 (SEQ ID NO:5), or human NELL2 (SEQ ID NO:7), rat NELL2 (SEQ ID NO:9), mouse NELL2 (SEQ ID NO:11), chicken NELL2 (SEQ ID NO:13). In some embodiments, the nucleic acid sequence can also include sequences such as those with substantial sequence similarity, such as sequences having at least about 75% sequence similarity with any portion of the sequences listed above.

Further the nucleic acid can include an expression vector for expressing the nucleic acid sequence encoding a NELL peptide, such as NELL1 or NELL2 peptide. For example, the expression vector can be pIZT/V5-His (Invitrogen), and selective markers can also include blastcidin and neomycin.

Such expression systems can include a carrier such as a viral carrier or viral vector, peptide carrier or a short polymer molecule.

Nucleic acid constructs can comprise expression and cloning vectors should contain a selection gene, also termed a selectable marker, such as a gene that encodes a protein necessary for the survival or growth of a host cell transformed with the vector. The presence of this gene ensures that any host cell which deletes the vector will not obtain an advantage in growth or reproduction over transformed hosts. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate or tetracycline, (b) complement auxotrophic deficiencies.

Further, the nucleic acid sequence can also include additional nucleic acids which encode reporter products to monitor levels of gene expression, or encode peptide tags which can be visualized using known methods in the art to monitor levels of peptide expression. Additional sequences can be selected so as to not interfere with the expression of the nucleic acid, or the functionality of the expressed peptide product.

A promoter is recognized by the host organism and is operatively linked to the NELL encoding nucleic acid. Promoters are untranslated sequences located upstream from the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of nucleic acid under their control, including inducible and constitutive promoters. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g. the presence or absence of a nutrient or a change in temperature. At this time a large number of promoters recognized by a variety of potential host cells are well known.

A nucleic acid can be operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operatively linked to DNA for a polypeptide if it is expressed as a preprotein which participates in the secretion of the polypeptide; a promoter or enhancer is operatively linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to facilitate translation.

A NELL peptide can be expressed in any biological system. For example, a NELL peptide can be expressed in a bacterial system, a yeast system, a plant system or animal system.

In some embodiments, a NELL peptide can be expressed in a cell free expression system well known to those in the art. For example, E coli cell-free protein translation systems or wheat germ cell-free protein translation systems.

In some embodiments, a NELL peptide can be expressed in transgenic plant cell systems derived from tobacco, com, rice or soybean.

In some embodiments, a NELL peptide can be expressed in insect cells. The NELL1 and NELL2 peptides expressed in an insect system are functional forms of the protein.

COS7 cells can be used to produce NELL1 and NELL2 proteins at low levels, such as about 10 micrograms per litter medium, but require serum-containing medium for the expression. As for the signal peptides, NELL1 and NELL2 endogenous signal peptides permit expression in COS7 cells.

In one embodiment, the invention includes a method of expressing a functional NELL peptide, such as NELL1 or NELL2 peptide, using an insect cell line. In one embodiment, the insect cell can be a high five cell, Sf9 and other Sf cells.

In one embodiment, the invention can include a nucleic acid construct for expressing a NELL peptide, such as NELL1 and/or NELL2 peptide in an insect cell. The nucleic acid sequence can be a cDNA or genomic DNA, encoding at least a functional portion of a NELL peptide. For example, the nucleic acid sequence can be selected from the group including, but not limited to human NELL1 (SEQ ID NO:1), rat NELL1 (SEQ ID NO:3), mouse NELL1 (SEQ ID NO:5), or human NELL2 (SEQ ID NO:7), rat NELL2 (SEQ ID NO:9), mouse NELL2 (SEQ ID NO:11), chicken NELL2 (SEQ ID NO:13). The nucleic acid sequence can also include sequences such as those with substantial sequence similarity, such as sequences having at least about 75% sequence similarity with any portion of the sequences listed above.

In one embodiment, the invention can include a nucleic acid construct for expressing a NELL peptide, such as NELL1 and/or NELL2 peptide in a mammalian cell such as a Chinese hamster ovary cell (CHO cell). The nucleic acid sequence can be a cDNA or genomic DNA, encoding at least a functional portion of a NELL peptide. For example, the nucleic acid sequence can be selected from the group including, but not limited to human NELL1 (SEQ ID NO:1), rat NELL1 (SEQ ID NO:3), mouse NELL1 (SEQ ID NO:5), or human NELL2 (SEQ ID NO:7), rat NELL2 (SEQ ID NO:9), mouse NELL2 (SEQ ID NO:11), chicken NELL2 (SEQ ID NO:13). In some embodiments, the nucleic acid sequence can also include sequences such as those with substantial sequence similarity, such as sequences having at least about 75% sequence similarity with any portion of the sequences listed above.

In one embodiment, the invention can include cells that express functional NELL peptides. For example, the cell can be a CHO cell. In one embodiment, the cell can be transfected with a nucleic acid construct encoding a NELL peptide. For example, the cell line can be transfected transiently or stably with the nucleic acid construct encoding a NELL peptide. In one embodiment, NELL expressing nucleic acids (e.g., cDNA(s) can be cloned into gene expression vector or viral particles that are competent to transfect cells (such as insect cells or Chinese hamster ovary cells (CHO cells)).

The nucleic acid construct can also include a nucleic acid sequence encoding a NELL peptide, such as NELL1 or NELL2 peptide, in frame with a nucleic acid sequence encoding a non-insect signal peptide that is a non-insect secretory signal peptide.

In one embodiment, the invention can include cells that express functional NELL peptides, and can secrete functional proteins.

In one embodiment, the invention can include a polypeptide (amino acid sequence) comprising a NELL peptide, such as NELL1 or NELL2 peptide, and can include a non-insect secretory signal peptide.

Non-insect Secretory Signal Peptide

Useful non-insect secretory signal peptide for use in a NELL peptide expression system (e.g., a mammalian cell such as a CHO cell) can be any secretory signal peptide. Such non-insect secretory signal peptide can be, for example, plant secretory signal peptide, or animal secretory signal peptide, e.g., mammalian secretory signal peptide such as human secretory signal peptide. Various secretory signal peptide can be found at various publicly accessible sources, e.g., http.//proline.bic.nus.edu..sg/spdb/download.html.

Human secretory signal peptides are well documented. Examples of human secretory signal peptides include, but are not limited to,

1A02_human secretory signal peptide, 1A11_human secretory signal peptide, 1A25_human secretory signal peptide, 1A26_human secretory signal peptide, 1A30_human secretory signal peptide, 1A31_human secretory signal peptide, 1A33_human secretory signal peptide, 1A68_human secretory signal peptide, 1A80_human secretory signal peptide, 1B07_human secretory signal peptide, 1B15_human secretory signal peptide, 1B37_human secretory signal peptide, 1B40_human secretory signal peptide, 1B47_human secretory signal peptide, 1B48_human secretory signal peptide, 1B57_human secretory signal peptide, 1B59_human secretory signal peptide, 1B78_human secretory signal peptide, 1C03_human secretory signal peptide, 1C04_human secretory signal peptide, 1C05_human secretory signal peptide, 1C07_human secretory signal peptide, 1C14_human secretory signal peptide, 1C15_human secretory signal peptide, 1C16_human secretory signal peptide, 1C17_human secretory signal peptide, 1C18_human secretory signal peptide, 2B11_human secretory signal peptide, 2B14_human secretory signal peptide, 2B17_human secretory signal peptide, 2B1A_human secretory signal peptide, 2B1B_human secretory signal peptide, 2B32_human secretory signal peptide, 2DOB_human secretory signal peptide, 7B2_human secretory signal peptide, A1AT_human secretory signal peptide, A2GL_human secretory signal peptide, A2MG_human secretory signal peptide, ABP1_human secretory signal peptide, ACET_human secretory signal peptide, ACHB_human secretory signal peptide, ACRE_human secretory signal peptide, ACRO_human secretory signal peptide, ADA32_human secretory signal peptide, ADIPO_human secretory signal peptide, ADML_human secretory signal peptide, AGAL_human secretory signal peptide, AGR2_human secretory signal peptide, AGR3_human secretory signal peptide, AMBP_human secretory signal peptide, AMTN_human secretory signal peptide, ANF_human secretory signal peptide, ANGI_human secretory signal peptide, ANGL3_human secretory signal peptide, ANGL7_human secretory signal peptide, ANGT_human secretory signal peptide, ANPRA_human secretory signal peptide, APOA2_human secretory signal peptide, APOA4_human secretory signal peptide, APOA_human secretory signal peptide, APOC1_human secretory signal peptide, APOC2_human secretory signal peptide, APOC3_human secretory signal peptide, APOD_human secretory signal peptide, APOE_human secretory signal peptide, APOH_human secretory signal peptide, APR3_human secretory signal peptide, ASM_human secretory signal peptide, ASPG_human secretory signal peptide, BAMBI_human secretory signal peptide, BASI_human secretory signal peptide, BGAL_human secretory signal peptide, BGLR_human secretory signal peptide, BOC_human secretory signal peptide, BPIL1_human secretory signal peptide, BPI_human secretory signal peptide, BT3A3_human secretory signal peptide, BTNL8_human secretory signal peptide, C16L2_human secretory signal peptide, C1QT5_human secretory signal peptide, C1QT6_human secretory signal peptide, C1R_human secretory signal peptide, C1S_human secretory signal peptide, C4BPA_human secretory signal peptide, CA187_human secretory signal peptide, CADM3_human secretory signal peptide, CAH9_human secretory signal peptide, CALCR_human secretory signal peptide, CALRL_human secretory signal peptide, CALR_human secretory signal peptide, CAP7_human secretory signal peptide, CART_human secretory signal peptide, CASA1_human secretory signal peptide, CASB_human secretory signal peptide, CASK_human secretory signal peptide, CATC_human secretory signal peptide, CATE_human secretory signal peptide, CATG_human secretory signal peptide, CATW_human secretory signal peptide, CBG_human secretory signal peptide, CBLN3_human secretory signal peptide, CBLN4_human secretory signal peptide, CBPA1_human secretory signal peptide, CBPA3_human secretory signal peptide, CBPB1_human secretory signal peptide, CBPN_human secretory signal peptide, CCL11_human secretory signal peptide, CCL15_human secretory signal peptide, CCL19_human secretory signal peptide, CCL1_human secretory signal peptide, CCL22_human secretory signal peptide, CCL24_human secretory signal peptide, CCL2_human secretory signal peptide, CCL4_human secretory signal peptide, CCL5_human secretory signal peptide, CCL7_human secretory signal peptide, CD180_human secretory signal peptide, CD1A_human secretory signal peptide, CD244_human secretory signal peptide, CD276_human secretory signal peptide, CD27_human secretory signal peptide, CD28_human secretory signal peptide, CD2_human secretory signal peptide, CD320_human secretory signal peptide, CD34_human secretory signal peptide, CD3E_human secretory signal peptide, CD3G_human secretory signal peptide, CD3Z_human secretory signal peptide, CD45_human secretory signal peptide, CD5L_human secretory signal peptide, CD5_human secretory signal peptide, CD83_human secretory signal peptide, CD8B_human secretory signal peptide, CD99_human secretory signal peptide, CEAM1_human secretory signal peptide, CER1_human secretory signal peptide, CERU_human secretory signal peptide, CETP_human secretory signal peptide, CF126_human secretory signal peptide, CFAB_human secretory signal peptide, CFAH_human secretory signal peptide, CFAI_human secretory signal peptide, CH3L1_human secretory signal peptide, CH3L2_human secretory signal peptide, CHIT1_human secretory signal peptide, CL3L1_human secretory signal peptide, CLC11_human secretory signal peptide, CLC14_human secretory signal peptide, CLM1_human secretory signal peptide, CLM9_human secretory signal peptide, CLUS_human secretory signal peptide, CMA1_human secretory signal peptide, CMGA_human secretory signal peptide, CO1A1_human secretory signal peptide, CO2_human secretory signal peptide, CO3_human secretory signal peptide, CO4A2_human secretory signal peptide, CO5A2_human secretory signal peptide, CO6A1_human secretory signal peptide, CO6_human secretory signal peptide, CO7_human secretory signal peptide, CO9A1_human secretory signal peptide, CO9_human secretory signal peptide, COGA1_human secretory signal peptide, COLI_human secretory signal peptide, COL_human secretory signal peptide, CR2_human secretory signal peptide, CRDL2_human secretory signal peptide, CREG1_human secretory signal peptide, CRHBP_human secretory signal peptide, CRIM1_human secretory signal peptide, CRIS1_human secretory signal peptide, CRLF1_human secretory signal peptide, CRP_human secretory signal peptide, CSF2R_human secretory signal peptide, CSF2_human secretory signal peptide, CSF3R_human secretory signal peptide, CSPG2_human secretory signal peptide, CST9F_human secretory signal peptide, CST9_human secretory signal peptide, CTHR1_human secretory signal peptide, CTRB1_human secretory signal peptide, CXCL7_human secretory signal peptide, CXL10_human secretory signal peptide, CYTC_human secretory signal peptide, CYTL1_human secretory signal peptide, CYTN_human secretory signal peptide, CYTS_human secretory signal peptide, CYTT_human secretory signal peptide, D103A_human secretory signal peptide, DB127_human secretory signal peptide, DCD_human secretory signal peptide, DEF1_human secretory signal peptide, DKK1_human secretory signal peptide, DKK3_human secretory signal peptide, DKK4_human secretory signal peptide, DLK_human secretory signal peptide, DLL4_human secretory signal peptide, DNAS1_human secretory signal peptide, ECP_human secretory signal peptide, EDAR_human secretory signal peptide, EFNB1_human secretory signal peptide, EFNB3_human secretory signal peptide, EGFL8_human secretory signal peptide, EGFR_human secretory signal peptide, EGLN_human secretory signal peptide, ELA2A_human secretory signal peptide, ELA2B_human secretory signal peptide, ELAF_human secretory signal peptide, ENPL_human secretory signal peptide, ENPP7_human secretory signal peptide, EPGN_human secretory signal peptide, EPHB1_human secretory signal peptide, EPHB6_human secretory signal peptide, EPOR_human secretory signal peptide, EPO_human secretory signal peptide, ESAM_human secretory signal peptide, EST1_human secretory signal peptide, F13B_human secretory signal peptide, FA11_human secretory signal peptide, FA5_human secretory signal peptide, FA8_human secretory signal peptide, FCGR1_human secretory signal peptide, FCGRN_human secretory signal peptide, FCN1_human secretory signal peptide, FCN3_human secretory signal peptide, FCRL2_human secretory signal peptide, FCRLA_human secretory signal peptide, FETUA_human secretory signal peptide, FGF19_human secretory signal peptide, FGF21_human secretory signal peptide, FGF23_human secretory signal peptide, FGFR3_human secretory signal peptide, FGFR4_human secretory signal peptide, FGRL1_human secretory signal peptide, FIBA_human secretory signal peptide, FIBG_human secretory signal peptide, FKB14_human secretory signal peptide, FKBP2_human secretory signal peptide, FLRT2_human secretory signal peptide, FSHB_human secretory signal peptide, FSTL1_human secretory signal peptide, FSTL3_human secretory signal peptide, FST_human secretory signal peptide, FZD3_human secretory signal peptide, G6B_human secretory signal peptide, GALC_human secretory signal peptide, GDN_human secretory signal peptide, GELS_human secretory signal peptide, GI24_human secretory signal peptide, GLHA_human secretory signal peptide, GLPA_human secretory signal peptide, GLPB_human secretory signal peptide, GLPE_human secretory signal peptide, GLUC_human secretory signal peptide, GNS_human secretory signal peptide, GP1BA_human secretory signal peptide, GPI8_human secretory signal peptide, GPIX_human secretory signal peptide, GPR56_human secretory signal peptide, GPR97_human secretory signal peptide, GRAB_human secretory signal peptide, GREM1_human secretory signal peptide, GROA_human secretory signal peptide, GRP78_human secretory signal peptide, GRP_human secretory signal peptide, HA22_human secretory signal peptide, HA23_human secretory signal peptide, HA25_human secretory signal peptide, HA27_human secretory signal peptide, HB21_human secretory signal peptide, HB23_human secretory signal peptide, HB24_human secretory signal peptide, HB25_human secretory signal peptide, HB2B_human secretory signal peptide, HB2C_human secretory signal peptide, HB2K_human secretory signal peptide, HEP2_human secretory signal peptide, HGFA_human secretory signal peptide, HIS1_human secretory signal peptide, HIS3_human secretory signal peptide, HLAE_human secretory signal peptide, HPSE_human secretory signal peptide, HPT_human secretory signal peptide, HV103_human secretory signal peptide, HV303_human secretory signal peptide, I10R1_human secretory signal peptide, I10R2_human secretory signal peptide, I17RA_human secretory signal peptide, I17RB_human secretory signal peptide, I17RC_human secretory signal, I18BP_human secretory signal peptide, I20RA_human secretory signal peptide, I20RB_human secretory signal peptide, I22RA_human secretory signal peptide, IBP1 human secretory signal peptide, IBP2 human secretory signal peptide, IBP3_human secretory signal peptide, IBP7_human secretory signal peptide, IFN16_human secretory signal peptide, IFNA2_human secretory signal peptide, IFNA4_human secretory signal peptide, IFNA5_human secretory signal peptide, IFNA6_human secretory signal peptide, IFNA7_human secretory signal peptide, IFNK_human secretory signal peptide, IFNW1_human secretory signal peptide, IGF2_human secretory signal peptide, IGFL1_human secretory signal peptide, IGFL3_human secretory signal peptide, IL10_human secretory signal peptide, IL12A_human secretory signal peptide, IL12B_human secretory signal peptide, IL17F_human secretory signal peptide, IL17_human secretory signal peptide, IL19_human secretory signal peptide, IL1R1_human secretory signal peptide, IL1RA_human secretory signal peptide, IL20_human secretory signal peptide, IL21R_human secretory signal peptide, IL22_human secretory signal peptide, IL24_human secretory signal peptide, IL25_human secretory signal peptide, IL2RA_human secretory signal peptide, IL2RB_human secretory signal peptide, IL2RG_human secretory signal peptide, IL2_human secretory signal peptide, IL3_human secretory signal peptide, IL4_human secretory signal peptide, IL5RA_human secretory signal peptide, IL6RA_human secretory signal peptide , IL7RA_human secretory signal peptide, IL7_human secretory signal peptide, IL9_human secretory signal peptide, ILRL1_human secretory signal peptide, INAR2_human secretory signal peptide, INHA_human secretory signal peptide, INSL3_human secretory signal peptide, INSL4_human secretory signal peptide, INSL5_human secretory signal peptide, INS_human secretory signal peptide, IPSP_human secretory signal peptide, IRBP_human secretory signal peptide, ISK6_human secretory signal peptide, ITA2B_human secretory signal peptide, ITA2_human secretory signal peptide, ITA3_human secretory signal peptide, ITA4_human secretory signal peptide, ITA6_human secretory signal peptide, ITA7_human secretory signal peptide, ITAE_human secretory signal peptide, ITAL_human secretory signal peptide, ITAV_human secretory signal peptide, ITAX_human secretory signal peptide, ITB2_human secretory signal peptide, ITB4_human secretory signal peptide, ITIH4_human secretory signal peptide, JAM1_human secretory signal peptide, JAM2_human secretory signal peptide, JAM3_human secretory signal peptide, JAML1_human secretory signal peptide, KAZD1_human secretory signal peptide, KI2L1_human secretory signal peptide, KIRR2_human secretory signal peptide, KLK3_human secretory signal peptide, KLKB1_human secretory signal peptide, KNG1_human secretory signal peptide, KTEL1_human secretory signal peptide, KV403_human secretory signal peptide, KV404_human secretory signal peptide, L1CAM_human secretory signal peptide, LALBA_human secretory signal peptide, LAMB1_human secretory signal peptide, LAMC1_human secretory signal peptide, LAMP1_human secretory signal peptide, LAMP2_human secretory signal peptide, LBP_human secretory signal peptide, LCAT_human secretory signal peptide, LCN1_human secretory signal peptide, LCTL_human secretory signal peptide, LEUK_human secretory signal peptide, LG3BP_human secretory signal peptide, LIF_human secretory signal peptide, LIPG_human secretory signal peptide, LIPP_human secretory signal peptide, LIRA3_human secretory signal peptide, LMAN1_human secretory signal peptide, LPH_human secretory signal peptide, LRC55_human secretory signal peptide, LRRN1_human secretory signal peptide, LSHB_human secretory signal peptide, LUM_human secretory signal peptide, LU_human secretory signal peptide, LV605_human secretory signal peptide, LY86_human secretory signal peptide, LYAM2_human secretory signal peptide, LYPA3_human secretory signal peptide, LYPD6_human secretory signal peptide, LYSC_human secretory signal peptide, MBL2_human secretory signal peptide, MCP_human secretory signal peptide, MFAP4_human secretory signal peptide, MGP_human secretory signal peptide, MIA_human secretory signal peptide, MIME_human secretory signal peptide, MIP2A_human secretory signal peptide, MIP2B_human secretory signal peptide, MK_human secretory signal peptide, MMP1_human secretory signal peptide, MOTI_human secretory signal peptide, MOX2R_human secretory signal peptide, MPRD_human secretory signal peptide, MPRI_human secretory signal peptide, MPZL3_human secretory signal peptide, MSMB_human secretory signal peptide, MYP0_human secretory signal peptide, NAGAB_human secretory signal peptide, NELL1_human secretory signal peptide, NELL2_human secretory signal peptide, NETO2_human secretory signal peptide, NEU1_human secretory signal peptide, NEU2_human secretory signal peptide, NGAL_human secretory signal peptide, NID1_human secretory signal peptide, NID2_human secretory signal peptide, NLGNX_human secretory signal peptide, NMB_human secretory signal peptide, NOV_human secretory signal peptide, NPTN_human secretory signal peptide, NPY_human secretory signal peptide, NRP1_human secretory signal peptide, NTRK2_human secretory signal peptide, NXPH3_human secretory signal peptide, OLFL1_human secretory signal peptide, OTOR_human secretory signal peptide, OXLA_human secretory signal peptide, P3IP1_human secretory signal peptide, PAHO_human secretory signal peptide, PARM1_human secretory signal peptide, PCDBA_human secretory signal peptide, PCOC2_human secretory signal peptide, PCYXL_human secretory signal peptide, PDGFA_human secretory signal peptide, PDIA1_human secretory signal peptide, PDIA3_human secretory signal peptide, PDYN_human secretory signal peptide, PEBP4_human secretory signal peptide, PECA1_human secretory signal peptide, PG12B_human secretory signal peptide, PGFRA_human secretory signal peptide, PGFRB_human secretory signal peptide, PGH1_human secretory signal peptide, PGRP2_human secretory signal peptide, PIGT_human secretory signal peptide, PIP_human secretory signal peptide, PLBL2_human secretory signal peptide, PLF4_human secretory signal peptide, PLOD1_human secretory signal peptide, PORIM_human secretory signal peptide, PPA6_human secretory signal peptide, PPAP_human secretory signal peptide, PPGB_human secretory signal peptide, PPIB_human secretory signal peptide, PRB4_human secretory signal peptide, PRLR_human secretory signal peptide, PRL_human secretory signal peptide, PROK1_human secretory signal peptide, PROK2_human secretory signal peptide, PROP_human secretory signal peptide, PROZ_human secretory signal peptide, PRP1_human secretory signal peptide, PRPC_human secretory signal peptide, PRRT3_human secretory signal peptide, PTGDS_human secretory signal peptide, PTHY_human secretory signal peptide, PTPRG_human secretory signal peptide, PYY_human secretory signal peptide, PZP_human secretory signal peptide, REG1A_human secretory signal peptide, REG3G_human secretory signal peptide, RIB1_human secretory signal peptide, RIB2_human secretory signal peptide, RISC_human secretory signal peptide, RNAS1_human secretory signal peptide, RNAS4_human secretory signal peptide, S39A6_human secretory signal peptide, SAA4_human secretory signal peptide, SAA_human secretory signal peptide, SAMP_human secretory signal peptide, SCG1_human secretory signal peptide, SCRG1_human secretory signal peptide, SEM4B_human secretory signal peptide, SEM6B_human secretory signal peptide, SEMG1_human secretory signal peptide, SEMG2_human secretory signal peptide, SEPP1_human secretory signal peptide, SFRP2_human secretory signal peptide, SFRP3_human secretory signal peptide, SFTPG_human secretory signal peptide, SG1D4_human secretory signal peptide, SG3A1_human secretory signal peptide, SHBG_human secretory signal peptide, SIAL_human secretory signal peptide, SIDT2_human secretory signal peptide, SLAF6_human secretory signal peptide, SLAF7_human secretory signal peptide, SLAF8_human secretory signal peptide, SLPI_human secretory signal peptide, SMR3B_human secretory signal peptide, SMS_human secretory signal peptide, SODE_human secretory signal peptide, SOSD1_human secretory signal peptide, SOST_human secretory signal peptide, SPIT1_human secretory signal peptide, SPIT2_human secretory signal peptide, SRCH_human secretory signal peptide, SRGN_human secretory signal peptide, STAT_human secretory signal peptide, STC1_human secretory signal peptide, TCO1_human secretory signal peptide, TCO2_human secretory signal peptide, TENA_human secretory signal peptide, TETN_human secretory signal peptide, TFF1_human secretory signal peptide, TFF3_human secretory signal peptide, TFPI1_human secretory signal peptide, TGFR2_human secretory signal peptide, THBG_human secretory signal peptide, THYG_human secretory signal peptide, TICN2_human secretory signal peptide, TIE1_human secretory signal peptide, TIE2_human secretory signal peptide, TIMP1_human secretory signal peptide, TIMP2_human secretory signal peptide, TIMP3_human secretory signal peptide, TINAL_human secretory signal peptide, TLR1_human secretory signal peptide, TLR3_human secretory signal peptide, TLR4_human secretory signal peptide, TLR5_human secretory signal peptide, TM2D1_human secretory signal peptide, TMIG2_human secretory signal peptide, TMM25_human secretory signal peptide, TMM46_human secretory signal peptide, TMM66_human secretory signal peptide, TMM9B_human secretory signal peptide, TNFB_human secretory signal peptide, TNR14_human secretory signal peptide, TNR16_human secretory signal peptide, TNR18_human secretory signal peptide, TNR19_human secretory signal peptide, TNR1B_human secretory signal peptide, TNR5_human secretory signal peptide, TNR6B_human secretory signal peptide, TNR8_human secretory signal peptide, TNR9_human secretory signal peptide, TPO_human secretory signal peptide, TPSNR_human secretory signal peptide, TPSN_human secretory signal peptide, TR10D_human secretory signal peptide, TR11B_human secretory signal peptide, TR19L_human secretory signal peptide, TRBM_human secretory signal peptide, TRFE_human secretory signal peptide, TRFL_human secretory signal peptide, TRY1_human secretory signal peptide, TRY2_human secretory signal peptide, TSHB_human secretory signal peptide, TSP1_human secretory signal peptide, TVA2_human secretory signal peptide, TVB2_human secretory signal peptide, TXD12_human secretory signal peptide, TXND4_human secretory signal peptide, TYRP1_human secretory signal peptide, UROK_human secretory signal peptide, UTER_human secretory signal peptide, UTS2_human secretory signal peptide, VCAM1_human secretory signal peptide, VEGFA_human secretory signal peptide, VEGFC_human secretory signal peptide, VGFR3_human secretory signal peptide, VMO1_human secretory signal peptide, VSIG2_human secretory signal peptide, VSIG4_human secretory signal peptide, VSTM1_human secretory signal peptide, VTNC_human secretory signal peptide, VWF_human secretory signal peptide, WISP2_human secretory signal peptide, X3CL1_human secretory signal peptide, XCL2_human secretory signal peptide, YK001_human secretory signal peptide, YQ001_human secretory signal peptide, ZA2G_human secretory signal peptide, ZG16_human secretory signal peptide, ZP2_human secretory signal peptide.

The sequences of these secretory signal peptides are as following:

1A02_HUMAN SPdb195 Homo sapiens (Human) (SEQ ID NO: 15) MAVMAPRTLVLLLSGALALTQTWA 1A11_HUMAN SPdb208 Homo sapiens (Human) (SEQ ID NO: 16) MAVMAPRTLLLLLSGALALTQTWA 1A25_HUMAN SPdb276 Homo sapiens (Human) (SEQ ID NO: 17) MAVMAPRTLVLLLSGALALTQTWA 1A26_HUMAN SPdb277 Homo sapiens (Human) (SEQ ID NO: 18) MAVMAPRTLVLLLSGALALTQTWA 1A30_HUMAN SPdb279 Homo sapiens (Human) (SEQ ID NO: 19) MAVMAPRTLLLLLSGALALTHTWA 1A31_HUMAN SPdb280 Homo sapiens (Human) (SEQ ID NO: 20) MAVMAPRTLLLLLLGALALTQTWA 1A33_HUMAN SPdb282 Homo sapiens (Human) (SEQ ID NO: 21) MAVMAPRTLLLLLLGALALTQTWA 1A68_HUMAN SPdb287 Homo sapiens (Human) (SEQ ID NO: 22) MAVMAPRTLVLLLSGALALTQTWA 1A80_HUMAN SPdb290 Homo sapiens (Human) (SEQ ID NO: 23) MAVMPPRTLLLLLSGALALTQTWA 1B07_HUMAN SPdb298 Homo sapiens (Human) (SEQ ID NO: 24) MLVMAPRTVLLLLSAALALTETWA IB15_HUMAN SPdb302 Homo sapiens (Human) (SEQ ID NO: 25) MRVTAPRTVLLLLSGALALTETWA 1B37_HUMAN SPdb306 Homo sapiens (Human) (SEQ ID NO: 26) MRVTAPRTLLLLLWGAVALTETWA 1B40_HUMAN SPdb309 Homo sapiens (Human) (SEQ ID NO: 27) MRVTAPRTLLLLLWGAVALTETWA 1B47_HUMAN SPdb315 Homo sapiens (Human) (SEQ ID NO: 28) MRVTAPRTLLLLLWGAVALTETWA 1B48_HUMAN SPdb316 Homo sapiens (Human) (SEQ ID NO: 29) MLVMAPRTVLLLLSAALALTETWA 1B57_HUMAN SPdb325 Homo sapiens (Human) (SEQ ID NO: 30) MRVTAPRTVLLLLWGAVALTETWA 1B59_HUMAN SPdb327 Homo sapiens (Human) (SEQ ID NO: 31) MRVTAPRTLLLLLWGALALTETWA 1B78_HUMAN SPdb330 Homo sapiens (Human) (SEQ ID NO: 32) MRVTAPRTVLLLLWGAVALTETWA 1C03_HUMAN SPdb340 Homo sapiens (Human) (SEQ ID NO: 33) MRVMAPRTLILLLSGALALTETWA 1C04_HUMAN SPdb342 Homo sapiens (Human) (SEQ ID NO: 34) MRVMAPRTLILLLSGALALTETWA 1C05_HUMAN SPdb343 Homo sapiens (Human) (SEQ ID NO: 35) MRVMAPRTLILLLSGALALTETWA 1C07_HUMAN SPdb345 Homo sapiens (Human) (SEQ ID NO: 36) MRVMAPRALLLLLSGGLALTETWA 1C14_HUMAN SPdb348 Homo sapiens (Human) (SEQ ID NO: 37) MRVMAPRTLILLLSGALALTETWA 1C15_HUMAN SPdb349 Homo sapiens (Human) (SEQ ID NO: 38) MRVMAPRTLLLLLSGALALTETWA 1C16_HUMAN SPdb350 Homo sapiens (Human) (SEQ ID NO: 39) MRVMAPRTLILLLSGALALTETWA 1C17_HUMAN SPdb351 Homo sapiens (Human) (SEQ ID NO: 40) MRVMAPQALLLLLSGALALIETWA 1C18_HUMAN SPdb352 Homo sapiens (Human) (SEQ ID NO: 41) MRVMAPRALLLLLSGGLALTETWA 2B11_HUMAN SPdb440 Homo sapiens (Human) (SEQ ID NO: 42) MVCLKLPGGSCMTALTVTLMVLSSPLALA 2B14_HUMAN SPdb441 Homo sapiens (Human) (SEQ ID NO: 43) MVCLKFPGGSCMAALTVTLMVLSSPLALA 2B17_HUMAN SPdb442 Homo sapiens (Human) (SEQ ID NO: 44) MVCLKLPGGSCMAALTVTLMVLSSPLALA 2B1A_HUMAN SPdb445 Homo sapiens (Human) (SEQ ID NO: 45) MVCLRLPGGSCMAVLTVTLMVLSSPLALA 2B1B_HUMAN SPdb446 Homo sapiens (Human) (SEQ ID NO: 46) MVCLRLPGGSCMAVLTVTLMVLSSPLALA 2B32_HUMAN SPdb449 Homo sapiens (Human) (SEQ ID NO: 47) MVCLKLPGGSSLAALTVTLMVLSSRLAFA 2DOB_HUMAN SPdb458 Homo sapiens (Human) (SEQ ID NO: 48) MGSGWVPWVVALLVNLTRLDSSMTQG 7B2_HUMAN SPdb1113 Homo sapiens (Human) (SEQ ID NO: 49) MVSRMVSTMLSGLLFWLASGWTPAFA A1AT_HUMAN SPdb1182 Homo sapiens (Human) (SEQ ID NO: 50) MPSSVSWGILLLAGLCCLVPVSLA A2GL_HUMAN SPdb1223 Homo sapiens (Human) (SEQ ID NO: 51) MSSWSRQRPKSPGGIQPHVSRTLFLLLLLAASAWG A2MG_HUMAN SPdb1225 Homo sapiens (Human) (SEQ ID NO: 52) MGKNKLLHPSLVLLLLVLLPTDA ABP1_HUMAN SPdb2047 Homo sapiens (Human) (SEQ ID NO: 53) MPALGWAVAAILMLQTAMA ACET_HUMAN SPdb2859 Homo sapiens (Human) (SEQ ID NO: 54) MGQGWATAGLPSLLFLLLCYGHPLLVPSQEA ACHB_HUMAN SPdb2984 Homo sapiens (Human) (SEQ ID NO: 55) MTPGALLMLLGALGAPLAPGVRG ACHE_HUMAN SPdb2999 Homo sapiens (Human) (SEQ ID NO: 56) MARAPLGVLLLLGLLGRGVG ACRO_HUMAN SPdb4311 Homo sapiens (Human) (SEQ ID NO: 57) MVEMLPTAILLVLAVSVVA ADA32_HUMAN SPdb5197 Homo sapiens (Human) (SEQ ID NO: 58) MFRLWLLLAGLCGLLA ADIPO_HUMAN SPdb5938 Homo sapiens (Human) (SEQ ID NO: 59) MLLLGAVLLLLALPGHDQ ADML_HUMAN SPdb5966 Homo sapiens (Human) (SEQ ID NO: 60) MKLVSVALMYLGSLAFLGADT AGAL_HUMAN SPdb6382 Homo sapiens (Human) (SEQ ID NO: 61) MQLRNPELHLGCALALRFLALVSWDIPGARA AGR2_HUMAN SPdb6560 Homo sapiens (Human) (SEQ ID NO: 62) MEKIPVSAFLLLVALSYTLA AGR3_HUMAN SPdb6563 Homo sapiens (Human) (SEQ ID NO: 63) MMLHSALGLCLLLVTVSSNLA AMBP_HUMAN SPdb8414 Homo sapiens (Human) (SEQ ID NO: 64) MRSLGALLLLLSACLAVSA AMTN_HUMAN SPdb9141 Homo sapiens (Human) (SEQ ID NO: 65) MRSTILLFCLLGSTRS ANF_HUMAN SPdb9494 Homo sapiens (Human) (SEQ ID NO: 66) MSSFSTTTVSFLLLLAFQLLGQTRA ANGI_HUMAN SPdb9521 Homo sapiens (Human) (SEQ ID NO: 67) MVMGLGVLLLVFVLGLGLTPPTLA ANGL3_HUMAN SPdb9540 Homo sapiens (Human) (SEQ ID NO: 68) MFTIKLLLFIVPLVIS ANGL7_HUMAN SPdb9551 Homo sapiens (Human) (SEQ ID NO: 69) MLKKPLSAVTWLCIFIVAFVSHPAWL ANGT_HUMAN SPdb9580 Homo sapiens (Human) (SEQ ID NO: 70) MRKRAPQSEMAPAGVSLRATILCLLAWAGLAAG ANPRA_HUMAN SPdb9903 Homo sapiens (Human) (SEQ ID NO: 71) MPGPRRPAGSRLRLLLLLLLPPLLLLLRGSHA APOA2_HUMAN SPdb10884 Homo sapiens (Human) (SEQ ID NO: 72) MKLLAATVLLLTICSLEG APOA4_HUMAN SPdb10891 Homo sapiens (Human) (SEQ ID NO: 73) MFLKAVVLTLALVAVAGARA APOA_HUMAN SPdb10900 Homo sapiens (Human) (SEQ ID NO: 74) MEHKEVVLLLLLFLKSAAP APOC1_HUMANSPdb10907 Homo sapiens (Human) (SEQ ID NO: 75) MRLFLSLPVLVVVLSIVLEGPAPAQG APOC2_HUMANSPdb10917 Homo sapiens (Human) (SEQ ID NO: 76) MGTRLLPALFLVLLVLGFEVQG APOC3_HUMANSPdb10924 Homo sapiens (Human) (SEQ ID NO: 77) MQPRVLLVVALLALLASARA APOD_HUMAN SPdb10936 Homo sapiens (Human) (SEQ ID NO: 78) MVMLLLLLSALAGLFGAAEG APOE_HUMAN SPdb10946 Homo sapiens (Human) (SEQ ID NO: 79) MKVLWAALLVTFLAGCQA APOH_HUMAN SPdb10966 Homo sapiens (Human) (SEQ ID NO: 80) MISPVLILFSSFLCHVAIA APR3_HUMAN SPdb11011 Homo sapiens (Human) (SEQ ID NO: 81) MAPHGPGSLTTLVPWAAALLLALGVERALA ASM_HUMAN SPdb16794 Homo sapiens (Human) (SEQ ID NO: 82) MPRYGASLRQSCPRSGREQGQDGTAGAPGLLWMGLVLALALALALA ASPG_HUMAN SPdb17050 Homo sapiens (Human) (SEQ ID NO: 83) MARKSNLPVLLVPFLLCQALVRC BAMBI_HUMAN SPdb23773 Homo sapiens (Human) (SEQ ID NO: 84) MDRHSSYIFIWLQLELCAMA BASI_HUMAN SPdb23850 Homo sapiens (Human) (SEQ ID NO: 85) MAAALFVLLGFALLGTHGASG BGAL_HUMAN SPdb24876 Homo sapiens (Human) (SEQ ID NO: 86) MPGFLVRILLLLLVLLLLGPTRG BGLR_HUMAN SPdb24971 Homo sapiens (Human) (SEQ ID NO: 87) MARGSAVAWAALGPLLWGCALG BOC_HUMAN SPdb25928 Homo sapiens (Human) (SEQ ID NO: 88) MLRGTMTAWRGMRPEVTLACLLLATAGCFA BPI_L1_HUMAN SPdb26090 Homo sapiens (Human) (SEQ ID NO: 89) MAWASRLGLLLALLLPVVGA BPI_HUMAN SPdb26097 Homo sapiens (Human) (SEQ ID NO: 90) MRENMARGPCNAPRWVSLMVLVAIGTAVTAA BT3A3_HUMAN SPdb26592 Homo sapiens (Human) (SEQ ID NO: 91) MKMASSLAFLLLNFHVSLFLVQLLTPCSA BTNL8_HUMANSPdb26692 Homo sapiens (Human) (SEQ ID NO: 92) MALMLSLVLSLLKLGSG C16L2_HUMAN SPdb27156 Homo sapiens (Human) (SEQ ID NO: 93) MEAPGPRALRTALCGGCCCLLLCAQLAVA C1QT5_HUMAN SPdb27240 Homo sapiens (Human) (SEQ ID NO: 94) MRPLLVLLLLGLAAG C1QT6_HUMAN SPdb27243 Homo sapiens (Human) (SEQ ID NO: 95) MVTAALGPVWAALLLFLLMCEIPMVEL C1R_HUMAN SPdb27257 Homo sapiens (Human) (SEQ ID NO: 96) MWLLYLLVPALFCRAGG C1S_HUMAN SPdb27262 Homo sapiens (Human) (SEQ ID NO: 97) MWCIVLFSLLAWVYA C4BPA_HUMANSPdb27346 Homo sapiens (Human) (SEQ ID NO: 98) MHPPKTPSGALHRKRKMAAWPFSRLWKVSDPILFQMTLIAALLPAVLG CA187_HUMAN SPdb27946 Homo sapiens (Human) (SEQ ID NO: 99) MAGPAIHTAPMLFLVLLLPLELSLA CADM3_HUMAN SPdb28390 Homo sapiens (Human) (SEQ ID NO: 100) MGAPAASLLLLLLLFACCWAPGGA CAH9_HUMAN SPdb28555 Homo sapiens (Human) (SEQ ID NO: 101) MAPLCPSPWLPLLIPAPAPGLTVQLLLSLLLLVPVHP CALCR_HUMAN SPdb28726 Homo sapiens (Human) (SEQ ID NO: 102) MRFTFTSRCLALFLLLNHPTPILP CALRL_HUMAN SPdb28899 Homo sapiens (Human) (SEQ ID NO: 103) MEKKCTLYFLVLLPFFMILVTA CALR_HUMAN SPdb28916 Homo sapiens (Human) (SEQ ID NO: 104) MLLSVPLLLGLLGLAVA CAP7_HUMAN SPdb29157 Homo sapiens (Human) (SEQ ID NO: 105) MTRLTVLALLAGLLASSRAGSSPLLD CART_HUMAN SPdb29961 Homo sapiens (Human) (SEQ ID NO: 106) MESSRVRLLPLLGAALLLMLPLLGTRA CASA1_HUMANSPdb29986 Homo sapiens (Human) (SEQ ID NO: 107) MRLLILTCLVAVALA CASB_HUMAN SPdb30005 Homo sapiens (Human) (SEQ ID NO: 108) MKVLILACLVALALA CASK_HUMAN SPdb30048 Homo sapiens (Human) (SEQ ID NO: 109) MKSFLLVVNALALTLPFLAV CATC_HUMAN SPdb30354 Homo sapiens (Human) (SEQ ID NO: 110) MGAGPSLLLAALLLLLSGDGAVRC CATE_HUMAN SPdb30381 Homo sapiens (Human) (SEQ ID NO: 111) MKTLLLLLLVLLELGEA CATG_HUMAN SPdb30393 Homo sapiens (Human) (SEQ ID NO: 112) MQPLLLLLAFLLPTGAEA CATW_HUMAN SPdb30490 Homo sapiens (Human) (SEQ ID NO: 113) MALTAHPSCLLALLVAGLAQG CBG_HUMAN SPdb30913 Homo sapiens (Human) (SEQ ID NO: 114) MPLLLYTCLLWLPTSGLWTVQA CBLN3_HUMANSPdb31375 Homo sapiens (Human) (SEQ ID NO: 115) MLGAKPHWLPGPLHSPGLPLVLVLLALGAGWA CBLN4_HUMANSPdb31377 Homo sapiens (Human) (SEQ ID NO: 116) MGSGRRALSAVPAVLLVLTLPGLPVWA CBPA1_HUMANSPdb31413 Homo sapiens (Human) (SEQ ID NO: 117) MRGLLVLSVLLGAVFG CBPA3_HUMANSPdb31420 Homo sapiens (Human) (SEQ ID NO: 118) MRLILPVGLIATTLA CBPB1_HUMANSPdb31459 Homo sapiens (Human) (SEQ ID NO: 119) MLALLVLVTVALASA CBPN_HUMAN SPdb31531 Homo sapiens (Human) (SEQ ID NO: 120) MSDLLSVFLHLLLLFKLVAP CCL11_HUMAN SPdb32635 Homo sapiens (Human) (SEQ ID NO: 121) MKVSAALLWLLLIAAAFSPQGLA CCL15_HUMAN SPdb32644 Homo sapiens (Human) (SEQ ID NO: 122) MKVSVAALSCLMLVAVLGSQA CCL19_HUMAN SPdb32652 Homo sapiens (Human) (SEQ ID NO: 123) MALLLALSLLVLWTSPAPTLS CCL1_HUMAN SPdb32654 Homo sapiens (Human) (SEQ ID NO: 124) MQIITTALVCLLLAGMWPEDVDS CCL22_HUMAN SPdb32663 Homo sapiens (Human) (SEQ ID NO: 125) MARLQTALLVVLVLLAVALQATEA CCL24_HUMAN SPdb32668 Homo sapiens (Human) (SEQ ID NO: 126) MAGLMTIVTSLLFLGVCAHHIIPTGS CCL2_HUMAN SPdb32684 Homo sapiens (Human) (SEQ ID NO: 127) MKVSAALLCLLLIAATFIPQGLA CCL4_HUMAN SPdb32700 Homo sapiens (Human) (SEQ ID NO: 128) MKLCVTVLSLLMLVAAFCSPALS CCL5_HUMAN SPdb32709 Homo sapiens (Human) (SEQ ID NO: 129) MKVSAAALAVILIATALCAPASA CCL7_HUMAN SPdb32717 Homo sapiens (Human) (SEQ ID NO: 130) MKASAALLCLLLTAAAFSPQGLA CD180_HUMAN SPdb33618 Homo sapiens (Human) (SEQ ID NO: 131) MAFDVSCFFWVVLFSAGCKVITS CD1A_HUMAN SPdb33624 Homo sapiens (Human) (SEQ ID NO: 132) MLFLLLPLLAVLPGDG CD244_HUMAN SPdb33672 Homo sapiens (Human) (SEQ ID NO: 133) MLGQVVTLILLLLLKVYQGKG CD276_HUMAN SPdb33680 Homo sapiens (Human) (SEQ ID NO: 134) MLRRRGSPGMGVHVGAALGALWFCLTGA CD27_HUMAN SPdb33684 Homo sapiens (Human) (SEQ ID NO: 135) MARPHPWWLCVLGTLVGLS CD28_HUMAN SPdb33689 Homo sapiens (Human) (SEQ ID NO: 136) MLRLLLALNLFPSIQVTG CD2_HUMAN SPdb33718 Homo sapiens (Human) (SEQ ID NO: 137) MSFPCKFVASFLLIFNVSSKGAVS CD320_HUMAN SPdb33725 Homo sapiens (Human) (SEQ ID NO: 138) MSGGWMAQVGAWRTGALGLALLLLLGLGLGLEAAA CD34_HUMAN SPdb33729 Homo sapiens (Human) (SEQ ID NO: 139) MLVRRGARAGPRMPRGWTALCLLSLLPSGFM CD3E_HUMAN SPdb33763 Homo sapiens (Human) (SEQ ID NO: 140) MQSGTHWRVLGLCLLSVGVWGQ CD3G_HUMAN SPdb33770 Homo sapiens (Human) (SEQ ID NO: 141) MEQGKGLAVLILAIILLQGTLA CD3Z_HUMAN SPdb33777 Homo sapiens (Human) (SEQ ID NO: 142) MKWKALFTAAILQAQLPITEA CD45_HUMAN SPdb33804 Homo sapiens (Human) (SEQ ID NO: 143) MYLWLKLLAFGFAFLDTEVFVTG CD5L_HUMAN SPdb33861 Homo sapiens (Human) (SEQ ID NO: 144) MALLFSLILAICTRPGFLA CD5_HUMAN SPdb33872 Homo sapiens (Human) (SEQ ID NO: 145) MPMGSLQPLATLYLLGMLVASCLG CD83_HUMAN SPdb33912 Homo sapiens (Human) (SEQ ID NO: 146) MSRGLQLLLLSCAYSLAPA CD8B_HUMAN SPdb33924 Homo sapiens (Human) (SEQ ID NO: 147) MRPRLWLLLAAQLTVLHGNSV CD99_HUMAN SPdb33932 Homo sapiens (Human) (SEQ ID NO: 148) MARGAALALLLFGLLGVLVAAP CEAM1_HUMAN SPdb34729 Homo sapiens (Human) (SEQ ID NO: 149) MGHLSAPLHRVRVPWQGLLLTASLLTFWNPPTTA CER1_HUMAN SPdb35232 Homo sapiens (Human) (SEQ ID NO: 150) MHLLLFQLLVLLPLGKT CERU_HUMAN SPdb35241 Homo sapiens (Human) (SEQ ID NO: 151) MKILILGIFLFLCSTPAWA CETP_HUMAN SPdb35298 Homo sapiens (Human) (SEQ ID NO: 152) MLAATVLTLALLGNAHA CF126_HUMAN SPdb35387 Homo sapiens (Human) (SEQ ID NO: 153) MAAALALVAGVLSGAVLPLWS CFAB_HUMAN SPdb35492 Homo sapiens (Human) (SEQ ID NO: 154) MGSNLSPQLCLMPFILGLLSGGVTT CFAH_HUMAN SPdb35506 Homo sapiens (Human) (SEQ ID NO: 155) MRLLAKIICLMLWAICVA CFAI_HUMAN SPdb35508 Homo sapiens (Human) (SEQ ID NO: 156) MKLLHVFLLFLCFHLRFC CH3L1_HUMAN SPdb36470 Homo sapiens (Human) (SEQ ID NO: 157) MGVKASQTGFVVLVLLQCCSA CH3L2_HUMAN SPdb36476 Homo sapiens (Human) (SEQ ID NO: 158) MGATTMDQKSLWAGVVVLLLLQGGSA CHIT1_HUMAN SPdb37892 Homo sapiens (Human) (SEQ ID NO: 159) MVRSVAWAGFMVLLMIPWGSA CL3L1_HUMAN SPdb39759 Homo sapiens (Human) (SEQ ID NO: 160) MQVSTAALAVLLCTMALCNQVLS CLC11_HUMAN SPdb39795 Homo sapiens (Human) (SEQ ID NO: 161) MQAAWLLGALVVPQLLGFGHG CLC14_HUMAN SPdb39798 Homo sapiens (Human) (SEQ ID NO: 162) MRPAFALCLLWQALWPGPGGG CLM1_HUMAN SPdb40173 Homo sapiens (Human) (SEQ ID NO: 163) MPLLTLYLLLFWLSGYSIA CLM9_HUMAN SPdb40190 Homo sapiens (Human) (SEQ ID NO: 164) MRLLVLLWGCLLLPGYEA CLUS_HUMAN SPdb41741 Homo sapiens (Human) (SEQ ID NO: 165) MMKTLLLFVGLLLTWESGQVLG CMA1_HUMAN SPdb41778 Homo sapiens (Human) (SEQ ID NO: 166) MLLLPLPLLLFLLCSRAEA CMGA_HUMAN SPdb41825 Homo sapiens (Human) (SEQ ID NO: 167) MRSAAVLALLLCAGQVTA CO1A1_HUMANSPdb42643 Homo sapiens (Human) (SEQ ID NO: 168) MFSFVDLRLLLLLAATALLTHG CO2_HUMAN SPdb42671 Homo sapiens (Human) (SEQ ID NO: 169) MGPLMVLFCLLFLYPGLADS CO3_HUMAN SPdb42683 Homo sapiens (Human) (SEQ ID NO: 170) MGPTSGPSLLLLLLTHLPLALG CO4A2_HUMANSPdb42702 Homo sapiens (Human) (SEQ ID NO: 171) MGRDQRAVAGPALRRWLLLGTVTVG CO5A2_HUMANSPdb42724 Homo sapiens (Human) (SEQ ID NO: 172) MMANWAEARPLLILIVLLGQFVSIKA CO6A1_HUMANSPdb42733 Homo sapiens (Human) (SEQ ID NO: 173) MRAARALLPLLLQACWTAA CO6_HUMAN SPdb42741 Homo sapiens (Human) (SEQ ID NO: 174) MARRSVLYFILLNALINKGQA CO7_HUMAN SPdb42748 Homo sapiens (Human) (SEQ ID NO: 175) MKVISLFILVGFIGEFQSFSSA CO9A1_HUMANSPdb42770 Homo sapiens (Human) (SEQ ID NO: 176) MKTCWKIPVFFFVCSFLEPWASA C09_HUMAN SPdb42781 Homo sapiens (Human) (SEQ ID NO: 177) MSACRSFAVAICILEISILTA COGA1_HUMAN SPdb44971 Homo sapiens (Human) (SEQ ID NO: 178) MWVSWAPGLWLLGLWATFGHG COLI_HUMAN SPdb45068 Homo sapiens (Human) (SEQ ID NO: 179) MPRSCCSRSGALLLALLLQASMEVRG COL_HUMAN SPdb45103 Homo sapiens (Human) (SEQ ID NO: 180) MEKILILLLVALSVAYA CR2_HUMAN SPdb47985 Homo sapiens (Human) (SEQ ID NO: 181) MGAAGLLGVFLALVAPGVLG CRDL2_HUMANSPdb48539 Homo sapiens (Human) (SEQ ID NO: 182) MVPEVRVLSSLLGLALLWFPLDSHA CREG1_HUMANSPdb48576 Homo sapiens (Human) (SEQ ID NO: 183) MAGLSRGSARALLAALLASTLLALLVSPARG CRHBP_HUMAN SPdb48678 Homo sapiens (Human) (SEQ ID NO: 184) MSPNFKLQCHFILIFLTALRGESR CRIM1_HUMANSPdb48688 Homo sapiens (Human) (SEQ ID NO: 185) MYLVAGDRGLAGCGHLLVSLLGLLLLLARSGTRA CRIS1_HUMAN SPdb48712 Homo sapiens (Human) (SEQ ID NO: 186) MEIKHLLFLVAAACLLPMLSM CRLF1_HUMAN SPdb48785 Homo sapiens (Human) (SEQ ID NO: 187) MPAGRRGPAAQSARRPPPLLPLLLLLCVLGAPRAGSG CRP_HUMAN SPdb48875 Homo sapiens (Human) (SEQ ID NO: 188) MEKLLCFLVLTSLSHAFG CSF2R_HUMAN SPdb49487 Homo sapiens (Human) (SEQ ID NO: 189) MLLLVTSLLLCELPHPAFLLIP CSF2_HUMAN SPdb49495 Homo sapiens (Human) (SEQ ID NO: 190) MWLQSLLLLGTVACSIS CSF3R_HUMAN SPdb49500 Homo sapiens (Human) (SEQ ID NO: 191) MARLGNCSLTWAALIILLLPGSLE CSPG2_HUMAN SPdb50031 Homo sapiens (Human) (SEQ ID NO: 192) MFINIKSILWMCSTLIVTHA CST9L_HUMAN SPdb50299 Homo sapiens (Human) (SEQ ID NO: 193) MLGLPWKGGLSWALLLLLLGSQILLIYA CST9_HUMAN SPdb50302 Homo sapiens (Human) (SEQ ID NO: 194) MSSPQRRKAMPWALSLLLMGFQLLVTYA CTHR1_HUMANSPdb50661 Homo sapiens (Human) (SEQ ID NO: 195) MRPQGPAASPQRLRGLLLLLLLQLPAPSSA CTRB1_HUMANSPdb50822 Homo sapiens (Human) (SEQ ID NO: 196) MAFLWLLSCWALLGTTFG CXCL7_HUMANSPdb52153 Homo sapiens (Human) (SEQ ID NO: 197) MSLRLDTTPSCNSARPLHALQVLLLLSLLLTALA CXL10_HUMAN SPdb52338 Homo sapiens (Human) (SEQ ID NO: 198) MNQTAILICCLIFLTLSGIQG CYTC_HUMAN SPdb56121 Homo sapiens (Human) (SEQ ID NO: 199) MAGPLRAPLLLLAILAVALAVSPAAG CYTL1_HUMANSPdb56131 Homo sapiens (Human) (SEQ ID NO: 200) MRTPGPLPVLLLLLAGAPAARP CYTN_HUMAN SPdb56136 Homo sapiens (Human) (SEQ ID NO: 201) MAQHLSTLLLLLATLAVALA CYTS_HUMAN SPdb56151 Homo sapiens (Human) (SEQ ID NO: 202) MARPLCTLLLLMATLAGALA CYTT_HUMAN SPdb56153 Homo sapiens (Human) (SEQ ID NO: 203) MAWPLCTLLLLLATQAVALA D103A_HUMAN SPdb56224 Homo sapiens (Human) (SEQ ID NO: 204) MRIHYLLFALLFLFLVPVPGHG DB127_HUMAN SPdb57844 Homo sapiens (Human) (SEQ ID NO: 205) MGLFMIIAILLFQKPTVTEQ DCD_HUMAN SPdb58564 Homo sapiens (Human) (SEQ ID NO: 206) MRFMTLLFLTALAGALVCA DEF1_HUMAN SPdb60113 Homo sapiens (Human) (SEQ ID NO: 207) MRTLAILAAILLVALQAQA DKK1_HUMAN SPdb62795 Homo sapiens (Human) (SEQ ID NO: 208) MMALGAAGATRVFVAMVAAALGGHPLLGVSA DKK3_HUMAN SPdb62800 Homo sapiens (Human) (SEQ ID NO: 209) MQRLGATLLCLLLAAAVPTAP DKK4_HUMAN SPdb62802 Homo sapiens (Human) (SEQ ID NO: 210) MVAAVLLGLSWLCSPLGA DLK_HUMAN SPdb62960 Homo sapiens (Human) (SEQ ID NO: 211) MTATEALLRVLLLLLAFGHSTYG DLL4_HUMAN SPdb62971 Homo sapiens (Human) (SEQ ID NO: 212) MAAASRSASGWALLLLVALWQQRAAG DNAS1_HUMAN SPdb64678 Homo sapiens (Human) (SEQ ID NO: 213) MRGMKLLGALLALAALLQGAVS ECP_HUMAN SPdb69940 Homo sapiens (Human) (SEQ ID NO: 214) MVPKLFTSQICLLLLLGLMGVEGSLHA EDAR_HUMAN SPdb70101 Homo sapiens (Human) (SEQ ID NO: 215) MAHVGDCTQTPWLPVLVVSLMCSARA EFNB1_HUMANSPdb71207 Homo sapiens (Human) (SEQ ID NO: 216) MARPGQRWLGKWLVAMVVWALCRLATP EFNB3_HUMAN SPdb71214 Homo sapiens (Human) (SEQ ID NO: 217) MGPPHSGPGGVRVGALLLLGVLGLVSG EGFL8_HUMAN SPdb72652 Homo sapiens (Human) (SEQ ID NO: 218) MGSRAELCTLLGGFSFLLLLIPGEG EGFR_HUMAN SPdb72662 Homo sapiens (Human) (SEQ ID NO: 219) MRPSGTAGAALLALLAALCPASRA EGLN_HUMAN SPdb72697 Homo sapiens (Human) (SEQ ID NO: 220) MDRGTLPLAVALLLASCSLSPTSLA ELA2A_HUMANSPdb73024 Homo sapiens (Human) (SEQ ID NO: 221) MIRTLLLSTLVAGALS ELA2B_HUMAN SPdb73028 Homo sapiens (Human) (SEQ ID NO: 222) MIRTLLLSTLVAGALS ELAF_HUMAN SPdb73040 Homo sapiens (Human) (SEQ ID NO: 223) MRASSFLIVVVFLIAGTLVLEA ENPL_HUMAN SPdb75253 Homo sapiens (Human) (SEQ ID NO: 224) MRALWVLGLCCVLLTFGSVRA ENPP7_HUMAN SPdb75282 Homo sapiens (Human) (SEQ ID NO: 225) MRGLAVLLTVALATLLAPGAG EPGN_HUMAN SPdb75639 Homo sapiens (Human) (SEQ ID NO: 226) MALGVPISVYLLFNAMTALTEE EPHB1_HUMANSPdb75671 Homo sapiens (Human) (SEQ ID NO: 227) MALDYLLLLLLASAVAA EPHB6_HUMANSPdb75686 Homo sapiens (Human) (SEQ ID NO: 228) MVCSLWVLLLVSSVLA EPOR_HUMAN SPdb75734 Homo sapiens (Human) (SEQ ID NO: 229) MDHLGASLWPQVGSLCLLLAGAAW EPO_HUMAN SPdb75747 Homo sapiens (Human) (SEQ ID NO: 230) MGVHECPAWLWLLLSLLSLPLGLPVLG ESAM_HUMAN SPdb76640 Homo sapiens (Human) (SEQ ID NO: 231) MISLPGPLVTNLLRFLFLGLSALAPPSRA EST1_HUMAN SPdb76818 Homo sapiens (Human) (SEQ ID NO: 232) MWLRAFILATLSASAAWG F13B_HUMAN SPdb78523 Homo sapiens (Human) (SEQ ID NO: 233) MRLKNLTFIIILIISGELYA FA11_HUMAN SPdb78712 Homo sapiens (Human) (SEQ ID NO: 234) MIFLYQVVHFILFTSVSG FA5_HUMAN SPdb78865 Homo sapiens (Human) (SEQ ID NO: 235) MFPGCPRLWVLVVLGTSWVGWGSQGTEA FA8_HUMAN SPdb78981 Homo sapiens (Human) (SEQ ID NO: 236) MQIELSTCFFLCLLRFCFS FCGR1_HUMANSPdb81611 Homo sapiens (Human) (SEQ ID NO: 237) MWFLTTLLLWVPVDG FCGRN_HUMAN SPdb81622 Homo sapiens (Human) (SEQ ID NO: 238) MGVPRPQPWALGLLLFLLPGSLG FCN1_HUMAN SPdb81643 Homo sapiens (Human) (SEQ ID NO: 239) MELSGATMARGLAVLLVLFLHIKNLPAQA FCN3_HUMAN SPdb81652 Homo sapiens (Human) (SEQ ID NO: 240) MDLLWILPSLWLLLLGGPACLKT FCRL2_HUMAN SPdb81673 Homo sapiens (Human) (SEQ ID NO: 241) MLLWSLLVIFDAVTEQADS FCRLA_HUMAN SPdb81677 Homo sapiens (Human) (SEQ ID NO: 242) MKLGCVLMAWALYLSLGVLWVAQMLLA FETUA_HUMAN SPdb82655 Homo sapiens (Human) (SEQ ID NO: 243) MKSLVLLLCLAQLWGCHS FGF19_HUMAN SPdb82742 Homo sapiens (Human) (SEQ ID NO: 244) MRSGCVVVHVWILAGLWLAVAGRP FGF21_HUMAN SPdb82761 Homo sapiens (Human) (SEQ ID NO: 245) MDSDETGFEHSGLWVSVLAGLLLGACQA FGF23_HUMAN SPdb82765 Homo sapiens (Human) (SEQ ID NO: 246) MLGARLRLWVCALCSVCSMSVLRA FGFR3_HUMAN SPdb82845 Homo sapiens (Human) (SEQ ID NO: 247) MGAPACALALCVAVAIVAGASS FGFR4_HUMAN SPdb82851 Homo sapiens (Human) (SEQ ID NO: 248) MRLLLALLGVLLSVPGPPVLS FGRL1_HUMAN SPdb82874 Homo sapiens (Human) (SEQ ID NO: 249) MTPSPLLLLLLPPLLLGAFPPAAA FIBA_HUMAN SPdb83006 Homo sapiens (Human) (SEQ ID NO: 250) MFSMRIVCLVLSVVGTAWT FIBG_HUMAN SPdb83075 Homo sapiens (Human) (SEQ ID NO: 251) MSWSLHPRNLILYFYALLFLSSTCVA FKB14_HUMAN SPdb83515 Homo sapiens (Human) (SEQ ID NO: 252) MRLFLWNAVLTLFVTSLIG FKBP2_HUMAN SPdb83570 Homo sapiens (Human) (SEQ ID NO: 253) MRLSWFRVLTVLSICLSAVAT FLRT2_HUMAN SPdb84949 Homo sapiens (Human) (SEQ ID NO: 254) MGLQTTKWPSHGAFFLKSWLIISLGLYSQVSKLLA FSHB_HUMAN SPdb87356 Homo sapiens (Human) (SEQ ID NO: 255) MKTLQFFFLFCCWKAICC FSTL1_HUMAN SPdb87403 Homo sapiens (Human) (SEQ ID NO: 256) MWKRWLALALALVAVAWVRA FSTL3_HUMAN SPdb87408 Homo sapiens (Human) (SEQ ID NO: 257) MRPGAPGPLWPLPWGALAWAVGFVSS FST_HUMAN SPdb87422 Homo sapiens (Human) (SEQ ID NO: 258) MVRARHQPGGLCLLLLLLCQFMEDRSAQA FZD3_HUMAN SPdb88844 Homo sapiens (Human) (SEQ ID NO: 259) MAMTWIVFSLWPLTVFMGHIGG G6B_HUMAN SPdb89267 Homo sapiens (Human) (SEQ ID NO: 260) MAVFLQLLPLLLSRAQG GALC_HUMAN SPdb90251 Homo sapiens (Human) (SEQ ID NO: 261) MTAAAGSAGRAAVPLLLCALLAPGGA GDN_HUMAN SPdb94028 Homo sapiens (Human) (SEQ ID NO: 262) MNWHLPLFLLASVTLPSIC GELS_HUMAN SPdb94075 Homo sapiens (Human) (SEQ ID NO: 263) MAPHRPAPALLCALSLALCALSLPVRA G124_HUMAN SPdb94473 Homo sapiens (Human) (SEQ ID NO: 264) MGVPTALEAGSWRWGSLLFALFLAASLGPVAA GLHA_HUMAN SPdb96435 Homo sapiens (Human) (SEQ ID NO: 265) MDYYRKYAAIFLVTLSVFLHVLHS GLPA_HUMAN SPdb98215 Homo sapiens (Human) (SEQ ID NO: 266) MYGKIIFVLLLSAIVSISA GLPB_HUMAN SPdb98238 Homo sapiens (Human) (SEQ ID NO: 267) MYGKIIFVLLLSEIVSISA GLPE_HUMAN SPdb98310 Homo sapiens (Human) (SEQ ID NO: 268) MYGKIIFVLLLSGIVSISA GLUC_HUMAN SPdb99110 Homo sapiens (Human) (SEQ ID NO: 269) MKSIYFVAGLFVMLVQGSWQ GNS_HUMAN SPdb100194 Homo sapiens (Human) (SEQ ID NO: 270) MRLLPLAPGRLRRGSPRHLPSCSPALLLLVLGGCLG GP1BA_HUMANSPdb100593 Homo sapiens (Human) (SEQ ID NO: 271) MPLLLLLLLLPSPLHP GPI8_HUMAN SPdb101405 Homo sapiens (Human) (SEQ ID NO: 272) MAVTDSLSRAATVLATVLLLSFGSVAA GPIX_HUMAN SPdb101411 Homo sapiens (Human) (SEQ ID NO: 273) MPAWGALFLLWATAEA GPR56_HUMAN SPdb102058 Homo sapiens (Human) (SEQ ID NO: 274) MTPQSLLQTTLFLLSLLFLVQGAHG GPR97_HUMAN SPdb102106 Homo sapiens (Human) (SEQ ID NO: 275) MATPRGLGALLLLLLLPTSG GRAB_HUMAN SPdb102370 Homo sapiens (Human) (SEQ ID NO: 276) MQPILLLLAFLLLPRADA GREM1_HUMAN SPdb102718 Homo sapiens (Human) (SEQ ID NO: 277) MSRTAYTVGALLLLLGTLLPAAEG GROA_HUMAN SPdb102897 Homo sapiens (Human) (SEQ ID NO: 278) MARAALSAAPSNPRLLRVALLLLLLVAAGRRAAG GRP78_HUMAN SPdb102967 Homo sapiens (Human) (SEQ ID NO: 279) MKLSLVAAMLLLLSAARA GRP_HUMAN SPdb103369 Homo sapiens (Human) (SEQ ID NO: 280) MRGSELPLVLLALVLCLAPRGRA HA22_HUMAN SPdb106653 Homo sapiens (Human) (SEQ ID NO: 281) MILNKALMLGALALTTVMSPCGG HA23_HUMAN SPdb106655 Homo sapiens (Human) (SEQ ID NO: 282) MILNKALMLGALALTTVMSPCGG HA25_HUMAN SPdb106658 Homo sapiens (Human) (SEQ ID NO: 283) MILNKALLLGALALTTVMSPCGG HA27_HUMAN SPdb106660 Homo sapiens (Human) (SEQ ID NO: 284) MILNKALMLGSLALTTVMSPCGG HB21_HUMAN SPdb106943 Homo sapiens (Human) (SEQ ID NO: 285) MSWKKALRIPGGLRAATVTLMLSMLSTPVAEG HB23_HUMAN SPdb106948 Homo sapiens (Human) (SEQ ID NO: 286) MSWKKALRIPGGLRVATVTLMLAMLSTSVAEG HB24_HUMAN SPdb106950 Homo sapiens (Human) (SEQ ID NO: 287) MSWKKALRIPGGLRVATVTLMLAMLSTPVAEG HB25_HUMAN SPdb106952 Homo sapiens (Human) (SEQ ID NO: 288) MSWKKALRIPGDLRVATVTLMLAMLSSLLAEG HB2B_HUMAN SPdb106955 Homo sapiens (Human) (SEQ ID NO: 289) MVCLRLPGGSCMAVLTVTLMVLSSPLALA HB2C_HUMAN SPdb106957 Homo sapiens (Human) (SEQ ID NO: 290) MVCLRLPGGSCMAVLTVTLMVLSSPLALA HB2K_HUMAN SPdb106966 Homo sapiens (Human) (SEQ ID NO: 291) MVCLKLPGGSCMAALTVTLTVLSSPLALA HEP2_HUMAN SPdb110024 Homo sapiens (Human) (SEQ ID NO: 292) MKHSLNALLIFLIITSAWG HGFA_HUMAN SPdb110611 Homo sapiens (Human) (SEQ ID NO: 293) MGRWAWVPSPWPPPGLGPFLLLLLLLLLLPRGFQP HIS1_HUMAN SPdb111008 Homo sapiens (Human) (SEQ ID NO: 294) MKFFVFALVLALMISMISA HIS3_HUMAN SPdb111571 Homo sapiens (Human) (SEQ ID NO: 295) MKFFVFALILALMLSMTGA HLAE_HUMAN SPdb113899 Homo sapiens (Human) (SEQ ID NO: 296) MVDGTLLLLLSEALALTQTWA HPSE_HUMAN SPdb115516 Homo sapiens (Human) (SEQ ID NO: 297) MLLRSKPALPPPLMLLLLGPLGPLSPGALPRPAQA HPT_HUMAN SPdb115541 Homo sapiens (Human) (SEQ ID NO: 298) MSALGAVIALLLWGQLFA HV103_HUMAN SPdb118845 Homo sapiens (Human) (SEQ ID NO: 299) MDWTWRILFLVAAATGAHS HV303_HUMAN SPdb118866 Homo sapiens (Human) (SEQ ID NO: 300) MEFGLSWLFLVAILKGVQC I10R1_HUMAN SPdb119663 Homo sapiens (Human) (SEQ ID NO: 301) MLPCLVVLLAALLSLRLGSDA I10R2_HUMAN SPdb119665 Homo sapiens (Human) (SEQ ID NO: 302) MAWSLGSWLGGCLLVSALG 117RA_HUMAN SPdb119686 Homo sapiens (Human) (SEQ ID NO: 303) MGAARSPPSAVPGPLLGLLLLLLGVLAPGGAS I17RB_HUMAN SPdb119688 Homo sapiens (Human) (SEQ ID NO: 304) MSLVLLSLAALCRSAVP I17RC_HUMAN SPdb119690 Homo sapiens (Human) (SEQ ID NO: 305) MPVPWFLLSLALGRSPVVLS I18BP_HUMAN SPdb119699 Homo sapiens (Human) (SEQ ID NO: 306) MRHNWTPDLSPLWVLLLCAHVVTLLVRA I20RA_HUMAN SPdb119707 Homo sapiens (Human) (SEQ ID NO: 307) MRAPGRPALRPLPLPPLLLLLLAAPWGRA I20RB_HUMAN SPdb119709 Homo sapiens (Human) (SEQ ID NO: 308) MQTFTMVLEEIWTSLFMWFFYALIPCLLT I22RA_HUMAN SPdb119711 Homo sapiens (Human) (SEQ ID NO: 309) MMPKHCFLGFLISFFLTGVAG IBP1_HUMAN SPdb119979 Homo sapiens (Human) (SEQ ID NO: 310) MSEVPVARVWLVLLLLTVQVGVTAG IBP2_HUMAN SPdbl 19988 Homo sapiens (Human) (SEQ ID NO: 311) MLPRVGCPALPLPPPPLLPLLPLLLLLLGASGGGGGARA IBP3_HUMAN SPdb119995 Homo sapiens (Human) (SEQ ID NO: 312) MQRARPTLWAAALTLLVLLRGPPVARA IBP7_HUMAN SPdb120015 Homo sapiens (Human) (SEQ ID NO: 313) MERPSLRALLLGAAGLLLLLLPLSSS IFN16_HUMAN SPdb122381 Homo sapiens (Human) (SEQ ID NO: 314) MALSFSLLMAVLVLSYKSICSLG IFNA2_HUMAN SPdb122393 Homo sapiens (Human) (SEQ ID NO: 315) MALTFALLVALLVLSCKSSCSVG IFNA4_HUMAN SPdb122399 Homo sapiens (Human) (SEQ ID NO: 316) MALSFSLLMAVLVLSYKSICSLG IFNA5_HUMAN SPdb122401 Homo sapiens (Human) (SEQ ID NO: 317) MALPFVLLMALVVLNCKSICS IFNA6_HUMAN SPdb122403 Homo sapiens (Human) (SEQ ID NO: 318) MALPFALLMALVVLSCKSSC IFNA7_HUMAN SPdb122405 Homo sapiens (Human) (SEQ ID NO: 319) MARSFSLLMVVLVLSYKSICSLG IFNK_HUMAN SPdb122481 Homo sapiens (Human) (SEQ ID NO: 320) MSTKPDMIQKCLWLEILMGIFIAGTLS IFNW1_HUMANSPdb122503 Homo sapiens (Human) (SEQ ID NO: 321) MALLFPLLAALVMTSYSPVGS IGF2_HUMAN SPdb122603 Homo sapiens (Human) (SEQ ID NO: 322) MGIPMGKSMLVLLTFLAFASCCIA IGFE1_HUMAN SPdb122611 Homo sapiens (Human) (SEQ ID NO: 323) MAPRGCIVAVFAIFCISRLLCSHG IGFL3_HUMAN SPdb122613 Homo sapiens (Human) (SEQ ID NO: 324) MRPRCCILALCVWITVFLLQCSKG IL10_HUMAN SPdb123103 Homo sapiens (Human) (SEQ ID NO: 325) METSSALLCCLVLLTGVRA IL12A_HUMAN SPdb123133 Homo sapiens (Human) (SEQ ID NO: 326) MCPARSLLLVATLVLLDHLSLA IL12B_HUMAN SPdb123153 Homo sapiens (Human) (SEQ ID NO: 327) MCHQQLVISWFSLVFLASPLVA IL17F_HUMAN SPdb123200 Homo sapiens (Human) (SEQ ID NO: 328) MVKYLLLSILGLAFLSEAAA IL17_HUMAN SPdb123202 Homo sapiens (Human) (SEQ ID NO: 329) MTPGKTSLVSLLLLLSLEAIVKA IL19_HUMAN SPdb123221 Homo sapiens (Human) (SEQ ID NO: 330) MKLQCVSLWLLGTILILCSVDNHG IL1R1_HUMAN SPdb123285 Homo sapiens (Human) (SEQ ID NO: 331) MKVLLRLICFIALLISS IL1RA_HUMAN SPdb123295 Homo sapiens (Human) (SEQ ID NO: 332) MEICRGLRSHLITLLLFLFHSETIC IL20_HUMAN SPdb123302 Homo sapiens (Human) (SEQ ID NO: 333) MKASSLAFSLLSAAFYLLWTPSTG IL21R_HUMAN SPdb123304 Homo sapiens (Human) (SEQ ID NO: 334) MPRGWAAPLLLLLLQGGWG IL22_HUMAN SPdb123314 Homo sapiens (Human) (SEQ ID NO: 335) MAALQKSVSSFLMGTLATSCLLLLALLVQGGAA IL24_HUMAN SPdb123324 Homo sapiens (Human) (SEQ ID NO: 336) MNFQQRLQSLWTLARPFCPPLLATASQMQMVVLPCLGFTLLLWSQVSGAQ G IL25_HUMAN SPdb123327 Homo sapiens (Human) (SEQ ID NO: 337) MRERPRLGEDSSLISLFLQVVAFLAMVMGTHT IL2RA_HUMAN SPdb123341 Homo sapiens (Human) (SEQ ID NO: 338) MDSYLLMWGLLTFIMVPGCQA IL2RB_HUMAN SPdb123348 Homo sapiens (Human) (SEQ ID NO: 339) MAAPALSWRLPLLILLLPLATSWASA IL2RG_HUMAN SPdb123355 Homo sapiens (Human) (SEQ ID NO: 340) MLKPSLPFTSLLFLQLPLLGVG IL2_HUMAN SPdb123376 Homo sapiens (Human) (SEQ ID NO: 341) MYRMQLLSCIALSLALVTNS IL3_HUMAN SPdb123414 Homo sapiens (Human) (SEQ ID NO: 342) MSRLPVLLLLQLLVRPGLQ IL4_HUMAN SPdb123438 Homo sapiens (Human) (SEQ ID NO: 343) MGLTSQLLPPLFFLLACAGNFVHG IL5RA_HUMAN SPdb123452 Homo sapiens (Human) (SEQ ID NO: 344) MIIVAHVLLILLGATEILQA IL6RA_HUMAN SPdb123469 Homo sapiens (Human) (SEQ ID NO: 345) MLAVGCALLAALLAAPGAA IL7RA_HUMAN SPdb123503 Homo sapiens (Human) (SEQ ID NO: 346) MTILGTTFGMVFSLLQVVSG IL7_HUMAN SPdb123506 Homo sapiens (Human) (SEQ ID NO: 347) MFHVSFRYIFGLPPLILVLLPVASS IL9_HUMAN SPdb123527 Homo sapiens (Human) (SEQ ID NO: 348) MLLAMVLTSALLLCSVAG ILRL1_HUMAN SPdb123591 Homo sapiens (Human) (SEQ ID NO: 349) MGFWILAILTILMYSTAA INAR2_HUMAN SPdb124806 Homo sapiens (Human) (SEQ ID NO: 350) MLLSQNAFIFRSLNLVLMVYISLVFG INHA_HUMAN SPdb124863 Homo sapiens (Human) (SEQ ID NO: 351) MVLHLLLFLLLTPQGGHS INSL3_HUMAN SPdb125047 Homo sapiens (Human) (SEQ ID NO: 352) MDPRLPAWALVLLGPALVFA INSL4_HUMAN SPdb125053 Homo sapiens (Human) (SEQ ID NO: 353) MASLFRSYLPAIWLLLSQLLRESLA INSL5_HUMAN SPdb125056 Homo sapiens (Human) (SEQ ID NO: 354) MKGSIFTLFLFSVLFAISEVRS INS_HUMAN SPdb125119 Homo sapiens (Human) (SEQ ID NO: 355) MALWMRLLPLLALLALWGPDPAAA IPSP_HUMAN SPdb125761 Homo sapiens (Human) (SEQ ID NO: 356) MQLFLLLCLVLLSPQGASL IRBP_HUMAN SPdb126007 Homo sapiens (Human) (SEQ ID NO: 357) MMREWVLLMSVLLCGLA ISK6_HUMAN SPdb126513 Homo sapiens (Human) (SEQ ID NO: 358) MKLSGMFLLLSLALFCFLTGVFS ITA2B_HUMAN SPdb128319 Homo sapiens (Human) (SEQ ID NO: 359) MARALCPLQALWLLEWVLLLLGPCAAPPAWA ITA2_HUMAN SPdb128324 Homo sapiens (Human) (SEQ ID NO: 360) MGPERTGAAPLPLLLVLALSQGILNCCLA ITA3_HUMAN SPdb128328 Homo sapiens (Human) (SEQ ID NO: 361) MGPGPSRAPRAPRLMLCALALMVAAGGCVVSA ITA4_HUMAN SPdb128331 Homo sapiens (Human) (SEQ ID NO: 362) MFPTESAWLGKRGANPGPEAAVRETVMLLLCLGVPTGRP ITA6_HUMAN SPdb128340 Homo sapiens (Human) (SEQ ID NO: 363) MAAAGQLCLLYLSAGLLSRLGAA ITA7_HUMAN SPdb128342 Homo sapiens (Human) (SEQ ID NO: 364) MAGARSRDPWGASGICYLFGSLLVELLFSRAVA ITAE_HUMAN SPdb128352 Homo sapiens (Human) (SEQ ID NO: 365) MWLFHTLLCIASLALLAA ITAL_HUMAN SPdb128355 Homo sapiens (Human) (SEQ ID NO: 366) MKDSCITVMAMALLSGFFFFAPASS ITAV_HUMAN SPdb128362 Homo sapiens (Human) (SEQ ID NO: 367) MAFPPRRRLRLGPRGLPLLLSGLLLPLCRA ITAX_HUMAN SPdb128364 Homo sapiens (Human) (SEQ ID NO: 368) MTRTRAALLLFTALATSLG ITB2_HUMAN SPdb128379 Homo sapiens (Human) (SEQ ID NO: 369) MLGLRPPLLALVGLLSLGCVLS ITB4_HUMAN SPdb128387 Homo sapiens (Human) (SEQ ID NO: 370) MAGPRPSPWARLLLAALISVSLSGTLA ITIH4_HUMAN SPdb128480 Homo sapiens (Human) (SEQ ID NO: 371) MKPPRPVRTCSKVLVLLSLLAIHQTTTA JAM1_HUMAN SPdb128758 Homo sapiens (Human) (SEQ ID NO: 372) MGTKAQVERKLLCLFILAILLCSLALG JAM2_HUMAN SPdb128761 Homo sapiens (Human) (SEQ ID NO: 373) MARRSRHRLLLLLLRYLVVALGYHKAYG JAM3_HUMAN SPdb128763 Homo sapiens (Human) (SEQ ID NO: 374) MALRRPPRLRLCARLPDFFLLLLFRGCLIGA JAML1_HUMANSPdb128766 Homo sapiens (Human) (SEQ ID NO: 375) MFCPLKLILLPVLLDYSLG KAZD1_HUMAN SPdb130734 Homo sapiens (Human) (SEQ ID NO: 376) MLPPPRPAAALALPVLLLLLVVLTPPPTGA KI2L1_HUMAN SPdb132984 Homo sapiens (Human) (SEQ ID NO: 377) MSLLVVSMACVGFFLLQGAWP KIRR2_HUMAN SPdb133175 Homo sapiens (Human) (SEQ ID NO: 378) MLRMRVPALLVLLFCFRGRA KLK3_HUMAN SPdb133637 Homo sapiens (Human) (SEQ ID NO: 379) MWVPVVFLTLSVTWIGA KLKB1_HUMAN SPdb133654 Homo sapiens (Human) (SEQ ID NO: 380) MILFKQATYFISLFATVSC KNG1_HUMAN SPdb133744 Homo sapiens (Human) (SEQ ID NO: 381) MKLITILFLCSRLLLSLT KTEE1_HUMANSPdb135048 Homo sapiens (Human) (SEQ ID NO: 382) MEWWASSPLRLWLLLFLLPSAQG KV403_HUMAN SPdb135854 Homo sapiens (Human) (SEQ ID NO: 383) MVLQTQVFISLLLWISGAYG KV404_HUMAN SPdb135855 Homo sapiens (Human) (SEQ ID NO: 384) MVLQTQVFISLLLWISGAYG L1CAM_HUMAN SPdb135937 Homo sapiens (Human) (SEQ ID NO: 385) MVVALRYVWPLLLCSPCLL LALBA_HUMAN SPdb136381 Homo sapiens (Human) (SEQ ID NO: 386) MRFFVPLFLVGILFPAILA LAMB1_HUMAN SPdb136411 Homo sapiens (Human) (SEQ ID NO: 387) MGLLQLLAFSFLALCRARVRA LAMC1_HUMAN SPdb136476 Homo sapiens (Human) (SEQ ID NO: 388) MRGSHRAAPALRPRGRLWPVLAVLAAAAAAGCA LAMP1_HUMAN SPdb136491 Homo sapiens (Human) (SEQ ID NO: 389) MAPRSARRPLLLLLPVAAARPHALSSA LAMP2_HUMAN SPdb136496 Homo sapiens (Human) (SEQ ID NO: 390) MVCFRLFPVPGSGLVLVCLVLGAVRSYA LBP_HUMAN SPdb136757 Homo sapiens (Human) (SEQ ID NO: 391) MGALARALPSILLALLLTSTPEALG LCAT_HUMAN SPdb136799 Homo sapiens (Human) (SEQ ID NO: 392) MGPPGSPWQWVTLLLGLLLPPAAP LCN1_HUMAN SPdb136934 Homo sapiens (Human) (SEQ ID NO: 393) MKPLLLAVSLGLIAALQA LCTL_HUMAN SPdb137082 Homo sapiens (Human) (SEQ ID NO: 394) MKPVWVATLLWMLLLVPRLGA LEUK_HUMAN SPdb139543 Homo sapiens (Human) (SEQ ID NO: 395) MATLLLLLGVLVVSPDALG LG3BP_HUMANSPdb140022 Homo sapiens (Human) (SEQ ID NO: 396) MTPPRLFWVWLLVAGTQG LIF_HUMAN SPdb140701 Homo sapiens (Human) (SEQ ID NO: 397) MKVLAAGVVPLLLVLHWKHGAG LIPG_HUMAN SPdb141547 Homo sapiens (Human) (SEQ ID NO: 398) MWLLLTMASLISVLGTTHG LIPP_HUMAN SPdb141584 Homo sapiens (Human) (SEQ ID NO: 399) MLPLWTLSLLLGAVAG LIRA3_HUMAN SPdb141630 Homo sapiens (Human) (SEQ ID NO: 400) MTPILTVLICLGLSLDPRTHVQA LMAN1_HUMAN SPdb141882 Homo sapiens (Human) (SEQ ID NO: 401) MAGSRQRGLRARVRPLFCALLLSLGRFVRG LPH_HUMAN SPdb142734 Homo sapiens (Human) (SEQ ID NO: 402) MELSWHVVFIALLSFSCWG LRC55_HUMAN SPdb144275 Homo sapiens (Human) (SEQ ID NO: 403) MGSLQHCCCLLPKMGDTWAQLPWPGPPHPAMLLISLLLAAGLMEISDA LRRN1_HUMANSPdb144478 Homo sapiens (Human) (SEQ ID NO: 404) MARMSFVIAACQLVLGLLMTSLTES LSHB_HUMAN SPdb144551 Homo sapiens (Human) (SEQ ID NO: 405) MEMLQGLLLLLLLSMGGAWA LUM_HUMAN SPdb145084 Homo sapiens (Human) (SEQ ID NO: 406) MSLSAFTLFLALIGGTSG LU_HUMAN SPdb145367 Homo sapiens (Human) (SEQ ID NO: 407) MEPPDAPAQARGAPRLLLLAVLLAAHPDAQA LV605_HUMAN SPdb145410 Homo sapiens (Human) (SEQ ID NO: 408) MAWAPLLLTLLAHCTDCWA LY86_HUMAN SPdb145461 Homo sapiens (Human) (SEQ ID NO: 409) MKGFTATLFLWTLIFPSCSG LYAM2_HUMAN SPdb145485 Homo sapiens (Human) (SEQ ID NO: 410) MIASQFLSALTLVLLIKESGA LYPA3_HUMANSPdb145568 Homo sapiens (Human) (SEQ ID NO: 411) MGLHLRPYRVGLLPDGLLFLLLLLMLLADPALP LYPD6_HUMANSPdb145583 Homo sapiens (Human) (SEQ ID NO: 412) MEPGPALAWLLLLSLLADCLKA LYSC_HUMAN SPdb145736 Homo sapiens (Human) (SEQ ID NO: 413) MKALIVLGLVLLSVTVQG MBL2_HUMAN SPdb148469 Homo sapiens (Human) (SEQ ID NO: 414) MSLFPSLPLLLLSMVAASYS MCP_HUMAN SPdb149004 Homo sapiens (Human) (SEQ ID NO: 415) MEPPGRRECPFPSWRFPGLLLAAMVLLLYSFSDA MFAP4_HUMAN SPdb152514 Homo sapiens (Human) (SEQ ID NO: 416) MKALLALPLLLLLSTPPCAPQ MGP_HUMAN SPdb152777 Homo sapiens (Human) (SEQ ID NO: 417) MKSLILLAILAALAVVTLC MIA_HUMAN SPdb153344 Homo sapiens (Human) (SEQ ID NO: 418) MARSLVCLGVIILLSAFSGPGVRG MIME_HUMAN SPdb153448 Homo sapiens (Human) (SEQ ID NO: 419) MKTLQSTLLLLLLVPLIKPA MIP2A_HUMAN SPdb153896 Homo sapiens (Human) (SEQ ID NO: 420) MARATLSAAPSNPRLLRVALLLLLLVAASRRAAG MIP2B_HUMAN SPdb153898 Homo sapiens (Human) (SEQ ID NO: 421) MAHATLSAAPSNPRLLRVALLLLLLVAASRRAAG MK_HUMAN SPdb154137 Homo sapiens (Human) (SEQ ID NO: 422) MQHRGFLLLTLLALLALTSA MMP1_HUMAN SPdb154439 Homo sapiens (Human) (SEQ ID NO: 423) MHSFPPLLLLLFWGVVSHS MOTI_HUMAN SPdb155899 Homo sapiens (Human) (SEQ ID NO: 424) MVSRKAVAALLVVHVAAMLASQTEA MOX2R_HUMAN SPdb155983 Homo sapiens (Human) (SEQ ID NO: 425) MLCPWRTANLGLLLILTIFLVAASSSLC MPRD_HUMAN SPdb156360 Homo sapiens (Human) (SEQ ID NO: 426) MFPFYSCWRTGLLLLLLAVAVRESWQ MPRI_HUMAN SPdb156380 Homo sapiens (Human) (SEQ ID NO: 427) MGAAAGRSPHLGPAPARRPQRSLLLLQLLLLVAAPGSTQA MPZL3_HUMANSPdb156425 Homo sapiens (Human) (SEQ ID NO: 428) MQQRGAAGSRGCALFPLLGVLFFQGVYIVFS MSMB_HUMAN SPdb158422 Homo sapiens (Human) (SEQ ID NO: 429) MNVLLGSVVIFATFVTLCNA MYP0_HUMAN SPdb164385 Homo sapiens (Human) (SEQ ID NO: 430) MAPGAPSSSPSPILAVLLFSSLVLSPAQA NAGAB_HUMAN SPdb165267 Homo sapiens (Human) (SEQ ID NO: 431) MLLKTVLLLGHVAQVLM NELL2_HUMAN SPdb168581 Homo sapiens (Human) (SEQ ID NO: 432) MESRVLLRTFCLIFGLGAVWG NETO2_HUMANSPdb168760 Homo sapiens (Human) (SEQ ID NO: 433) MALERLCSVLKVLLITVLVVEG NEU1_HUMAN SPdb168777 Homo sapiens (Human) (SEQ ID NO: 434) MAGPSLACCLLGLLALTSA NEU2_HUMAN SPdb168794 Homo sapiens (Human) (SEQ ID NO: 435) MPDTMLPACFLGLLAFSSA NGAL_HUMAN SPdb169201 Homo sapiens (Human) (SEQ ID NO: 436) MPLGLLWLGLALLGALHAQA NID1_HUMAN SPdb169544 Homo sapiens (Human) (SEQ ID NO: 437) MLASSSRIRAAWTRALLLPLLLAGPVGC NID2_HUMAN SPdb169547 Homo sapiens (Human) (SEQ ID NO: 438) MEGDRVAGRPVLSSLPVLLLLQLLMLRAAA NLGNX_HUMAN SPdb170305 Homo sapiens (Human) (SEQ ID NO: 439) MSRPQGLLWLPLLFTPVCVMLNSNVLLWLTALAIKFTLIDS NMB_HUMAN SPdb170485 Homo sapiens (Human) (SEQ ID NO: 440) MARRAGGARMFGSLLLFALLAAGV NOV_HUMAN SPdb171434 Homo sapiens (Human) (SEQ ID NO: 441) MQSVQSTSFCLRKQCLCLTFLLLHLLGQVAA NPTN_HUMAN SPdb171815 Homo sapiens (Human) (SEQ ID NO: 442) MSGSSLPSALALSLLLVSGSLLPGPGAA NPY_HUMAN SPdb171875 Homo sapiens (Human) (SEQ ID NO: 443) MLGNKRLGLSGLTLALSLLVCLGALAEA NRP1_HUMAN SPdb172978 Homo sapiens (Human) (SEQ ID NO: 444) MERGLPLLCAVLALVLAPAGA NTRK2_HUMAN SPdb173863 Homo sapiens (Human) (SEQ ID NO: 445) MSSWIRWHGPAMARLWGFCWLVVGFWRAAFA NXPH3_HUMAN SPdb177361 Homo sapiens (Human) (SEQ ID NO: 446) MQLTRCCFVFLVQGSLYLVICG OFFF1_HUMAN SPdb178511 Homo sapiens (Human) (SEQ ID NO: 447) MMVALRGASALLVLFLAAFLPPPQCTQD OTOR_HUMAN SPdb180733 Homo sapiens (Human) (SEQ ID NO: 448) MARILLLFLPGLVAVCA OXLA_HUMAN SPdb181241 Homo sapiens (Human) (SEQ ID NO: 449) MAPLALHLLVLVPILLSLVAS P3IP1_HUMAN SPdb181543 Homo sapiens (Human) (SEQ ID NO: 450) MLLAWVQAFLVSNMLLAEAYG PAHO_HUMAN SPdb182446 Homo sapiens (Human) (SEQ ID NO: 451) MAAARLCLSLLLLSTCVALLLQPLLGAQG PARM1_HUMAN SPdb184104 Homo sapiens (Human) (SEQ ID NO: 452) MVYKTLFALCILTAGWRVQS PCDBA_HUMAN SPdb184878 Homo sapiens (Human) (SEQ ID NO: 453) MAVRELCFPRQRQVLFLFLFWGVSLA PCOC2_HUMANSPdb185137 Homo sapiens (Human) (SEQ ID NO: 454) MRGANAWAPLCLLLAAATQLSRQ PCYXL_HUMAN SPdb185474 Homo sapiens (Human) (SEQ ID NO: 455) MARAPPLLAALTALLAAAAAGG PDGFA_HUMAN SPdb185687 Homo sapiens (Human) (SEQ ID NO: 456) MRTLACLLLLGCGYLAHVLA PDIA1_HUMAN SPdb185732 Homo sapiens (Human) (SEQ ID NO: 457) MLRRALLCLAVAALVRA PDIA3_HUMAN SPdb185743 Homo sapiens (Human) (SEQ ID NO: 458) MRLRRLALFPGVALLLAAARLAAA PDYN_HUMAN SPdb186941 Homo sapiens (Human) (SEQ ID NO: 459) MAWQGLVLAACLLMFPSTTA PEBP4_HUMAN SPdb187068 Homo sapiens (Human) (SEQ ID NO: 460) MGWTMRLVTAALLLGLMMVVTG PECA1_HUMANSPdb187074 Homo sapiens (Human) (SEQ ID NO: 461) MQPRWAQGATMWLGVLLTLLLCSSLEG PG12B_HUMAN SPdb188887 Homo sapiens (Human) (SEQ ID NO: 462) MKLASGFLVLWLSLGGGLA PGFRA_HUMAN SPdb189004 Homo sapiens (Human) (SEQ ID NO: 463) MGTSHPAFLVLGCLLTGLSLILC PGFRB_HUMANSPdb189010 Homo sapiens (Human) (SEQ ID NO: 464) MRLPGAMPALALKGELLLLSLLLLLEPQISQG PGH1_HUMAN SPdb189021 Homo sapiens (Human) (SEQ ID NO: 465) MSRSLLLRFLLFLLLLPPLPVLL PGRP2_HUMAN SPdb189713 Homo sapiens (Human) (SEQ ID NO: 466) MAQGVLWILLGLLLWSDPGTA PIGT_HUMAN SPdb191467 Homo sapiens (Human) (SEQ ID NO: 467) MAAAMPLALLVLLLLGPGGWC PIP_HUMAN SPdb191771 Homo sapiens (Human) (SEQ ID NO: 468) MRLLQLLFRASPATLLLVLCLQLGANKA PLBL2_HUMAN SPdb192296 Homo sapiens (Human) (SEQ ID NO: 469) MVGQMYCYPGSHLARALTRALALALVLALLVGPFLSGLAGA PLF4_HUMAN SPdb192496 Homo sapiens (Human) (SEQ ID NO: 470) MSSAAGFCASRPGLLFLGLLLLPLVVAFASA PLOD1_HUMANSPdb192587 Homo sapiens (Human) (SEQ ID NO: 471) MRPLLLLALLGWLLLAEA PORIM_HUMANSPdb194575 Homo sapiens (Human) (SEQ ID NO: 472) MGLGARGAWAALLLGTLQVLALLGAA PPA6_HUMAN SPdb195079 Homo sapiens (Human) (SEQ ID NO: 473) MITGVFSMRLWTPVGVLTSLAYCLHQRRVALA PPAP_HUMAN SPdb195174 Homo sapiens (Human) (SEQ ID NO: 474) MRAAPLLLARAASLSLGFLFLLFFWLDRSVLA PPGB_HUMAN SPdb195573 Homo sapiens (Human) (SEQ ID NO: 475) MIRAAPPPLFLLLLLLLLLVSWASRGEA PPIB_HUMAN SPdb195662 Homo sapiens (Human) (SEQ ID NO: 476) MKVLLAAALIAGSVFFLLLPGPSAA PRB4_HUMAN SPdb196833 Homo sapiens (Human) (SEQ ID NO: 477) MLLILLSVALLALSSA PRLR_HUMAN SPdb197321 Homo sapiens (Human) (SEQ ID NO: 478) MKENVASATVFTLLLFLNTCLLNG PRL_HUMAN SPdb197346 Homo sapiens (Human) (SEQ ID NO: 479) MNIKGSPWKGSLLLLLVSNLLLCQSVAP PROK1_HUMANSPdb198420 Homo sapiens (Human) (SEQ ID NO: 480) MRGATRVSIMLLLVTVSDC PROK2_HUMANSPdb198424 Homo sapiens (Human) (SEQ ID NO: 481) MRSLCCAPLLLLLLLPPLLLTPRAGDA PROP_HUMAN SPdb198449 Homo sapiens (Human) (SEQ ID NO: 482) MITEGAQAPRLLLPPLLLLLTLPATGS PROZ_HUMAN SPdb198539 Homo sapiens (Human) (SEQ ID NO: 483) MAGCVPLLQGLVLVLALHRVEPS PRP1_HUMAN SPdb198570 Homo sapiens (Human) (SEQ ID NO: 484) MLLILLSVALLALSSA PRPC_HUMAN SPdb198723 Homo sapiens (Human) (SEQ ID NO: 485) MLLILLSVALLAFSSA PRRT3_HUMAN SPdb198854 Homo sapiens (Human) (SEQ ID NO: 486) MASSPWGCVCGLLLLLLPLLGTGPALG PTGDS_HUMANSPdb203865 Homo sapiens (Human) (SEQ ID NO: 487) MATHHTLWMGLALLGVLGDLQA PTHY_HUMAN SPdb203993 Homo sapiens (Human) (SEQ ID NO: 488) MIPAKDMAKVMIVMLAICFLTKSDG PTPRG_HUMANSPdb204842 Homo sapiens (Human) (SEQ ID NO: 489) MRRLLEPCWWILFLKITSS PYY_HUMAN SPdb210515 Homo sapiens (Human) (SEQ ID NO :490) MVFVRRPWPALTTVLLALLVCLGALVDA PZP_HUMAN SPdb210532 Homo sapiens (Human) (SEQ ID NO: 491) MRKDRLLHLCLVLLLILLSASDSNS REG1A_HUMAN SPdb217426 Homo sapiens (Human) (SEQ ID NO: 492) MAQTSSYFMLISCLMFLSQSQG REG3G_HUMAN SPdb217443 Homo sapiens (Human) (SEQ ID NO: 493) MLPPMALPSVSWMLLSCLILLCQVQG RIB1_HUMAN SPdb220194 Homo sapiens (Human) (SEQ ID NO: 494) MEAPAAGLFLLLLLGTWAPAPGS RIB2_HUMAN SPdb220201 Homo sapiens (Human) (SEQ ID NO: 495) MAPPGSSTVFLLALTIIASTWA RISC_HUMAN SPdb221907 Homo sapiens (Human) (SEQ ID NO: 496) MELALRRSPVPRWLLLLPLLLGLNAG RNAS1_HUMANSPdb241351 Homo sapiens (Human) (SEQ ID NO: 497) MALEKSLVRLLLLVLILLVLGWVQPSLG RNAS4_HUMANSPdb241408 Homo sapiens (Human) (SEQ ID NO: 498) MALQRTHSLLLLLLLTLLGLGLVQPSYG S39A6_HUMAN SPdb264754 Homo sapiens (Human) (SEQ ID NO: 499) MARKLSVILILTFALSVTNPLHELKAAA SAA4_HUMAN SPdb264973 Homo sapiens (Human) (SEQ ID NO: 500) MRLFTGIVFCSLVMGVTS SAA_HUMAN SPdb264986 Homo sapiens (Human) (SEQ ID NO: 501) MKLLTGLVFCSLVLGVSS SAMP_HUMAN SPdb265378 Homo sapiens (Human) (SEQ ID NO: 502) MNKPLLWISVLTSLLEAFA SCG1_HUMAN SPdb266260 Homo sapiens (Human) (SEQ ID NO: 503) MQPTLLLSLLGAVGLAAVNS SRG1_HUMAN SPdb266759 Homo sapiens (Human) (SEQ ID NO: 504) MKLMVLVFTIGLTLLLGVQA SEM4B_HUMAN SPdb269059 Homo sapiens (Human) (SEQ ID NO: 505) MGLRSWLAAPWGALPPRPPLLLLLLLLLLLQPPPPTWA SEM6B_HUMAN SPdb269079 Homo sapiens (Human) (SEQ ID NO: 506) MQTPRASPPRPALLLLLLLLGGAHG SEMG1_HUMAN SPdb269090 Homo sapiens (Human) (SEQ ID NO: 507) MKPNIIFVLSLLLILEKQAAVMG SEMG2_HUMAN SPdb269095 Homo sapiens (Human) (SEQ ID NO: 508) MKSIILFVLSLLLILEKQAAVMG SEPP1_HUMAN SPdb269196 Homo sapiens (Human) (SEQ ID NO: 509) MWRSLGLALALCLLPSGGT SFRP2_HUMAN SPdb269743 Homo sapiens (Human) (SEQ ID NO: 510) MLQGPGSLLLLFLASHCCLGSARG SFRP3_HUMAN SPdb269747 Homo sapiens (Human) (SEQ ID NO: 511) MVCGSPGGMLLLRAGLLALAALCLLRVPGARA SFTPG_HUMAN SPdb269980 Homo sapiens (Human) (SEQ ID NO: 512) MGSGLPLVLLLTLLGSSHG SG1D4_HUMAN SPdb270016 Homo sapiens (Human) (SEQ ID NO: 513) MRLSVCLLMVSLALCCYQAHA SG3A1_HUMAN SPdb270025 Homo sapiens (Human) (SEQ ID NO: 514) MKLAALLGLCVALSCSSAAA SHBG_HUMAN SPdb270301 Homo sapiens (Human) (SEQ ID NO: 515) MESRGPLATSRLLLLLLLLLLRHTRQGWA SIAL_HUMAN SPdb270534 Homo sapiens (Human) (SEQ ID NO: 516) MKTALILLSILGMACA SIDT2_HUMAN SPdb270589 Homo sapiens (Human) (SEQ ID NO: 517) MFALGLPFLVLLVASVES SLAF6_HUMAN SPdb271065 Homo sapiens (Human) (SEQ ID NO: 518) MLWLFQSLLFVFCFGPGNVVS SLAF7_HUMAN SPdb271067 Homo sapiens (Human) (SEQ ID NO: 519) MAGSPTCLTLIYILWQLTGSAA SLAF8_HUMAN SPdb271069 Homo sapiens (Human) (SEQ ID NO: 520) MVMRPLWSLLLWEALLPITVTG SLPI_HUMAN SPdb271309 Homo sapiens (Human) (SEQ ID NO: 521) MKSSGLFPFLVLLALGTLAPWAVEG SMR3B_HUMAN SPdb271976 Homo sapiens (Human) (SEQ ID NO: 522) MKSLTWILGLWALAACFTPGES SMS_HUMAN SPdb272029 Homo sapiens (Human) (SEQ ID NO: 523) MLSCRLQCALAALSIVLALGCVTG SODE_HUMAN SPdb272720 Homo sapiens (Human) (SEQ ID NO: 524) MLALLCSCLLLAAGASDA SOSD1_HUMANSPdb273214 Homo sapiens (Human) (SEQ ID NO: 525) MLPPAIHFYLLPLACILMKSCLA SOST_HUMAN SPdb273220 Homo sapiens (Human) (SEQ ID NO: 526) MQLPLALCLVCLLVHTAFRVVEG SPIT1_HUMAN SPdb274564 Homo sapiens (Human) (SEQ ID NO: 527) MAPARTMARARLAPAGIPAVALWLLCTLGLQGTQA SPIT2_HUMAN SPdb274566 Homo sapiens (Human) (SEQ ID NO: 528) MAQLCGLRRSRAFLALLGSLLLSGVLA SRCH_HUMAN SPdb275436 Homo sapiens (Human) (SEQ ID NO: 529) MGHHRPWLHASVLWAGVASLLLPPAMTQ SRGN_HUMAN SPdb275551 Homo sapiens (Human) (SEQ ID NO: 530) MMQKLLKCSRLVLALALILVLESSVQG STAT_HUMAN SPdb277447 Homo sapiens (Human) (SEQ ID NO: 531) MKFLVFAFILALMVSMIGA STC1_HUMAN SPdb277483 Homo sapiens (Human) (SEQ ID NO: 532) MLQNSAVLLVLVISASA TCO1_HUMAN SPdb290388 Homo sapiens (Human) (SEQ ID NO: 533) MRQSHQLPLVGLLLFSFIPSQLC TCO2_HUMAN SPdb290391 Homo sapiens (Human) (SEQ ID NO: 534) MRHLGAFLFLLGVLGALT TENA_HUMAN SPdb291090 Homo sapiens (Human) (SEQ ID NO: 535) MGAMTQLLAGVFLAFLALATEG TETN_HUMAN SPdb291269 Homo sapiens (Human) (SEQ ID NO: 536) MELWGAYLLLCLFSLLTQVTT TFF1_HUMAN SPdb291574 Homo sapiens (Human) (SEQ ID NO: 537) MATMENKVICALVLVSMLALGTLA TFF3_HUMAN SPdb291583 Homo sapiens (Human) (SEQ ID NO: 538) MAARALCMLGLVLALLSSSSA TFPI1_HUMAN SPdb291601 Homo sapiens (Human) (SEQ ID NO: 539) MIYTMKKVHALWASVCLLLNLAPAPLNA TGFR2_HUMANSPdb291788 Homo sapiens (Human) (SEQ ID NO: 540) MGRGLLRGLWPLHIVLWTRIAS THBG_HUMAN SPdb292195 Homo sapiens (Human) (SEQ ID NO: 541) MSPFLYLVLLVLGLHATIHC THYG_HUMAN SPdb293853 Homo sapiens (Human) (SEQ ID NO: 542) MALVLEIFTLLASICWVSA TICN2_HUMAN SPdb293959 Homo sapiens (Human) (SEQ ID NO: 543) MRAPGCGRLVLPLLLLAAAALA TIE1_HUMAN SPdb293968 Homo sapiens (Human) (SEQ ID NO: 544) MVWRVPPFLLPILFLASHVGA TIE2_HUMAN SPdb293972 Homo sapiens (Human) (SEQ ID NO: 545) MDSLASLVLCGVSLLLSGTVEG TIMP1_HUMAN SPdb294965 Homo sapiens (Human) (SEQ ID NO: 546) MAPFEPLASGILLLLWLIAPSRA TIMP2_HUMAN SPdb294980 Homo sapiens (Human) (SEQ ID NO: 547) MGAAARTLRLALGLLLLATLLRPADA TIMP3_HUMAN SPdb294987 Homo sapiens (Human) (SEQ ID NO: 548) MTPWLGLIVLLGSWSLGDWGAEA TINAL_HUMAN SPdb295015 Homo sapiens (Human) (SEQ ID NO: 549) MWRCPLGLLLLLPLAGHLALG TLR1_HUMAN SPdb295471 Homo sapiens (Human) (SEQ ID NO: 550) MTSIFHFAIIFMLILQIRIQLSEE TLR3_HUMAN SPdb295485 Homo sapiens (Human) (SEQ ID NO: 551) MRQTEPCIYFWGGLLPFGMLCAS TLR4_HUMAN SPdb295493 Homo sapiens (Human) (SEQ ID NO: 552) MMSASRLAGTLIPAMAFLSCVRP TLR5_HUMAN SPdb295500 Homo sapiens (Human) (SEQ ID NO: 553) MGDHLDLLLGVVLMAGPVFG TM2D1_HUMAN SPdb295869 Homo sapiens (Human) (SEQ ID NO: 554) MAAAWPSGPSAPEAVTARLVGVLWFVSVTTGPWGAVA TMIG2_HUMANSPdb296142 Homo sapiens (Human) (SEQ ID NO: 555) MGSPGMVLGLLVQIWALQEASS TMM25_HUMAN SPdb296188 Homo sapiens (Human) (SEQ ID NO: 556) MALPPGPAALRHTLLLLPALLSSGWG TMM46_HUMAN SPdb296232 Homo sapiens (Human) (SEQ ID NO: 557) MWGARRSSVSSSWNAASLLQLLLAALLAAGARA TMM66_HUMAN SPdb296282 Homo sapiens (Human) (SEQ ID NO: 558) MAAACGPGAAGYCLLLGLHLFLLTAGPALG TMM9B_HUMAN SPdb296355 Homo sapiens (Human) (SEQ ID NO: 559) MATLWGGLLRLGSLLSLSCLALSVLLLAQLSDA TNFB_HUMAN SPdb296533 Homo sapiens (Human) (SEQ ID NO: 560) MTPPERLFLPRVCGTTLHLLLLGLLLVLLPGAQG TNR14_HUMAN SPdb296702 Homo sapiens (Human) (SEQ ID NO: 561) MEPPGDWGPPPWRSTPKTDVLRLVLYLTFLGAPCYAPA TNR16_HUMAN SPdb296704 Homo sapiens (Human) (SEQ ID NO: 562) MGAGATGRAMDGPRLLLLLLLGVSLGGA TNR18_HUMAN SPdb296709 Homo sapiens (Human) (SEQ ID NO: 563) MAQHGAMGAFRALCGLALLCALSLG TNR19_HUMAN SPdb296711 Homo sapiens (Human) (SEQ ID NO: 564) MALKVLLEQEKTFFTLLVLLGYLSCKVTC TNR1B_HUMANSPdb296718 Homo sapiens (Human) (SEQ ID NO: 565) MAPVAVWAALAVGLELWAAAHA TNR5_HUMAN SPdb296744 Homo sapiens (Human) (SEQ ID NO: 566) MVRLPLQCVLWGCLLTAVHP TNR6B_HUMANSPdb296751 Homo sapiens (Human) (SEQ ID NO: 567) MRALEGPGLSLLCLVLALPALLPVPAVRG TNR8_HUMAN SPdb296768 Homo sapiens (Human) (SEQ ID NO: 568) MRVLLAALGLLFLGALRA TNR9_HUMAN SPdb296771 Homo sapiens (Human) (SEQ ID NO: 569) MGNSCYNIVATLLLVLNFERTRS TPO_HUMAN SPdb298326 Homo sapiens (Human) (SEQ ID NO: 570) MELTELLLVVMLLLTARLTLS TPSNR_HUMANSPdb298428 Homo sapiens (Human) (SEQ ID NO: 571) MGTQEGWCLLLCLALSGA TPSN_HUMAN SPdb298434 Homo sapiens (Human) (SEQ ID NO: 572) MKSLSLLLAVALGLATAVSA TR10D_HUMAN SPdb298538 Homo sapiens (Human) (SEQ ID NO: 573) MGLWGQSVPTASSARAGRYPGARTASGTRPWLLDPKILKFVVFIVAVLLP VRVDS TR11B_HUMAN SPdb298559 Homo sapiens (Human) (SEQ ID NO: 574) MNKLLCCALVFLDISIKWTTQ TR19L_HUMAN SPdb298594 Homo sapiens (Human) (SEQ ID NO: 575) MKPSLLCRPLSCFLMLLPWPLATLT TRBM_HUMAN SPdb298866 Homo sapiens (Human) (SEQ ID NO: 576) MLGVLVLGALALAGLGFP TRFE_HUMAN SPdb299004 Homo sapiens (Human) (SEQ ID NO: 577) MRLAVGALLVCAVLGLCLA TRFL_HUMAN SPdb299019 Homo sapiens (Human) (SEQ ID NO: 578) MKLVFLVLLFLGALGLCLA TRY1_HUMAN SPdb303488 Homo sapiens (Human) (SEQ ID NO: 579) MNPLLILTFVAAALA TRY2_HUMAN SPdb303496 Homo sapiens (Human) (SEQ ID NO: 580) MNLLLILTFVAAAVA TSHB_HUMAN SPdb303727 Homo sapiens (Human) (SEQ ID NO: 581) MTALFLMSMLFGLACGQAMS TSP1_HUMAN SPdb303853 Homo sapiens (Human) (SEQ ID NO: 582) MGLAWGLGVLFLMEIVCGT TVA2_HUMAN SPdb304532 Homo sapiens (Human) (SEQ ID NO: 583) MAMLLGASVLILWLQPDWVNSQQKNDD TVB2_HUMAN SPdb304538 Homo sapiens (Human) (SEQ ID NO: 584) MGTSLLCWMALCLLGADHADT TXD12_HUMANSPdb304820 Homo sapiens (Human) (SEQ ID NO: 585) METRPRLGATCLLGFSFLLLVISSDG TXND4_HUMAN SPdb304996 Homo sapiens (Human) (SEQ ID NO: 586) MHPAVFLSLPDLRCSLLLLVTWVFTPVTT TYRP1_HUMANSPdb305383 Homo sapiens (Human) (SEQ ID NO: 587) MSAPKLLSLGCIFFPLLLFQQARA UROK_HUMAN SPdb311443 Homo sapiens (Human) (SEQ ID NO: 588) MRALLARLLLCVLVVSDSKG UTER_HUMAN SPdb311731 Homo sapiens (Human) (SEQ ID NO: 589) MKLAVTLTLVTLALCCSSASA UTS2_HUMAN SPdb311840 Homo sapiens (Human) (SEQ ID NO: 590) MYKLASCCLLFIGFLNPLLS VCAM1_HUMAN SPdb314239 Homo sapiens (Human) (SEQ ID NO: 591) MPGKMVVILGASNILWIMFAASQA VEGFA_HUMAN SPdb314841 Homo sapiens (Human) (SEQ ID NO: 592) MNFLLSWVHWSLALLLYLHHAKWSQA VEGFC_HUMAN SPdb314852 Homo sapiens (Human) (SEQ ID NO: 593) MHLLGFFSVACSLLAAALLPGPREAPAAAAA VGFR3_HUMAN SPdb315610 Homo sapiens (Human) (SEQ ID NO: 594) MQRGAALCLRLWLCLGLLDGLVSG VMO1_HUMAN SPdb317004 Homo sapiens (Human) (SEQ ID NO: 595) MERGAGAKLLPLLLLLRATGFTCA VSIG2_HUMAN SPdb318531 Homo sapiens (Human) (SEQ ID NO: 596) MAELPGPFLCGALLGFLCLSGLA VSIG4_HUMAN SPdb318533 Homo sapiens (Human) (SEQ ID NO: 597) MGILLGLLLLGHLTVDTYG VSTM1_HUMAN SPdb318698 Homo sapiens (Human) (SEQ ID NO: 598) MTAEFLSLLCLGLCLG VTNC_HUMAN SPdb318799 Homo sapiens (Human) (SEQ ID NO: 599) MAPLRPLLILALLAWVALA VWF_HUMAN SPdb318878 Homo sapiens (Human) (SEQ ID NO: 600) MIPARFAGVLLALALILPGTLC WISP2_HUMAN SPdb319573 Homo sapiens (Human) (SEQ ID NO: 601) MRGTPKTHLLAFSLLCLLSKVRT X3CL1_HUMAN SPdb320112 Homo sapiens (Human) (SEQ ID NO: 602) MAPISLSWLLRLATFCHLTVLLAG XCL2_HUMAN SPdb320139 Homo sapiens (Human) (SEQ ID NO: 603) MRLLILALLGICSLTAYIVEG YK001_HUMAN SPdb348747 Homo sapiens (Human) (SEQ ID NO: 604) MDSLRKMLISVAMLGAGAGVGYA YQ001_HUMAN SPdb352137 Homo sapiens (Human) (SEQ ID NO: 605) MGLPGLFCLAVLAASSFSKA ZA2G_HUMAN SPdb354335 Homo sapiens (Human) (SEQ ID NO: 606) MVPVLLSLLLLLGPAVP ZG16_HUMAN SPdb354803 Homo sapiens (Human) (SEQ ID NO: 607) MLTVALLALLCASASG ZP2_HUMAN SPdb355981 Homo sapiens (Human) (SEQ ID NO: 608) MACRQRGGSWSPSGWFNAGWSTYRSISLFFALVTSGNS

A preferred expression system using each specific human secretory signal peptide listed above is a CHO cell expression system.

In some embodiments, the human secretory signal peptide can include any combination of the aforelisted secretory signal peptide. In some embodiments, the human secretory signal peptide can specifically exclude any one or more of the aforelisted secretory signal peptide. For the sake of provding a concise description of the invention, listing of the above human secretory signal peptides is not repeated. Nonetheless this invention encompasses specific exclusion of any one of the human secretory signal peptide or nucleic acid sequences encoding each one of the secretory signal peptide listed above.

For the sake of providing a concise description of the invention, listing of nucleic acid sequences encoding each one of the secretory signal peptide listed above is omitted. Nonetheless, such nucleic acid sequences are publicly available and can be readily ascertained by a person of ordinary skill in the art. One public source of such nucleic acid sequence information is, e.g., http://proline.bic.nus.edu.sg/spdb/download.html.

In some embodiments, for mammalian expression systems, a protrypsin leading sequence can also be used.

In some embodiments, for production of NELL1 and/or NELL2 peptides in mammalian cells (e.g., CHO cells), the expression system for NELL1 and/or NELL2 can include the nucleic acid or cDNA that expresses an endogenous NELL signal peptide. In some embodiments, the method and system described herein specifically excludes an endogenous NELL signal peptide.

Peptide Purification

In some embodiments, the invention includes a method purifying NELL1 and/or NELL2 peptides secreted into culture media, according to standard peptide purification protocols, including, but not limited to those described below.

The method can also include collecting secreted NELL peptides and/or purifying NELL peptides for use. Peptide products can be tested for activity in a variety of functional or expression assays. For example in any assay, if a NELL peptide has a significant effect over a control substance on a given parameter, the NELL peptides can be functional to effect the measured parameter in a functional or expression assay.

In one embodiment, whether a selected cell expresses a selected nucleic acid sequence to express and/or secrete a NELL peptide can be examined. In one embodiment, the presence, amount or and/or activity of NELL peptides can be examined.

In one embodiment, NELL peptides detected and quantified by any of a number of methods well known to those of skill in the art. These can include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, western blotting, and the like.

In one embodiment, Western blot (immunoblot) analysis can be used to detect and quantify the presence of NELL peptide(s) in a selected sample. Western blot analysis is well known in the art.

The assays of this invention can be scored (as positive or negative or quantity of target polypeptide) according to standard methods well known to those of skill in the art. The particular method of scoring can depend on the assay format and choice of label. For example, a Western Blot assay can be scored by visualizing the colored product produced by an enzymatic label. A clearly visible colored band or spot at the correct molecular weight can be scored as a positive result, while the absence of a clearly visible spot or band can be scored as a negative. The intensity of the band or spot can provide a quantitative measure of target polypeptide concentration.

NELL peptides, when in non-monomeric forms can be a large molecule and many of the techniques that work for purification of smaller proteins may not work as well for NELL peptides. The unusually large size of NELL oligomeric protein molecules causes it to be incompatible with the industry standard techniques used to purify smaller proteins, such as monoclonal antibodies and blood clotting factors. Specifically, the industry standard media is made by cross-linking agarose (e.g., agarose columns) into round beads with dead-end channels. The channels are lined with a functional group and interaction of this functional group with a protein forms the basis for purification. Because the channels are dead-ends, a protein molecule enters the channels by the forces of diffusion. As the protein of interest increases in size, diffusion into the channel slows and purification efficiently drops.

Instead of traditional porous media (e.g., agarose columns), other media not traditionally used for proteins may be used to purify NELL peptides in a more efficient fashion both in terms of speed and amount of NELL peptides purified/recovered. The present invention provides a method for purifying NELL peptides with a higher productivity. A low productivity in purification of protein is generally considered a production rate limiting factor in protein production. Therefore achieving a higher productivity is important in protein production.

In some embodiments, the method includes subjecting crude NELL peptides to membrane chromatography to obtain purified NELL peptides. In some embodiments, the method includes using commercially available purification media in membrane format. In some embodiments, the NELL peptide is NELL-1 peptide. In some embodiments, the NELL peptide is NELL-2 peptide. By employing membrane purification media, an efficient purification process for the NELL peptide was developed whereas analogous efforts with porous media was much less efficient.

The ion-exchange membrane can be anion-exchange membrane or cation-exchange membrane. It is not at all obvious that membrane chromatography can be used to purify NELL peptides.

Further, it is particularly surprising that cation-exchange chromatography is effective for NELL peptide purification. For example, analysis of the NELL-1 primary protein sequence indicates that its theoretical p1 is 5.8. Under the pH ranges of 7-8.5 typically used to purify proteins, NELL-1 would be predicted to carry a net negative charge and therefore bind to anion exchange functional groups such as quaternary or secondary amines and not necessarily cation exchanges. Unexpectedly, however, NELL-1 is found to bind to both cation and anion exchange functional groups.

In some embodiments, purification of Nell-1 peptide/protein can be effected through a chromatography process using a medium including a metal cation bearing two, three or more charges, e.g., an earth metal cation, a rare earth metal cation. Examples of such metal ions are, e.g., Mg(++), Ca(++), Al(+++), and any rare earth ions Ln(+++). Such metal ions can be included in a buffer solution or eluting solution.

In some embodiments, purification can sequentially employ a cation exchange chromotography, an anion exchange chromotography and an hydroxyapatite medium. In other embodiments, it can also sequentially employ an anion exchange chromotography, a cation exchange chromotography and an hydroxyapatite medium. Alternatively, other combinations or steps can be introduced in between the aforementioned sequences.

The following examples are offered to illustrate, but not to limit the claimed invention.

EXAMPLES

In general, expression hosts can be bacteria, yeast and fungi, mammalian cells, plants, transgenic animals or it can also be cell-free expression systems such as those based on wheat germ or E. coli extracts. In general, expression elements can be Prokaryotic, Yeast, Mammalian and Plant promoters or viral promoters. Protein expression strategies can be: intra- or extracellular, fusion proteins and display strategies. Downstream processing of recombinant proteins can include: harvest, lysis, filtration, ultrafiltration, precipitation, and/or other protein processing/purification strategies that encompass protein capture, purification, polishing, and optimization.

Example 1. Expression of NELL Peptides

A cDNA fragment was ligated into the expression vector PiZT/V5-His (3.4 kb) (EcoRV site, Invitrogen) and included a melittin signal peptide, BamHI-EcoRI cDNA fragment of the mature rat NELL1 and a FLAG tag sequence. FIGS. 1A-1B are a depiction of the nucleic acid sequence of the cDNA construct used in this example, and corresponding predicted peptide sequence.

The High five cells (BTI-TN-5B1-4) were adapted to serum-free medium, and cells were transfected with the NELL1 peptide expression vector. Cells were treated with zeocin so as to select only cell populations expressing the NELL1 FLAG constructs. Surviving cell populations were confirmed to be stable transformants. Extracellular media was collected and tested for the presence of NELL1 peptide. NELL1 peptide was purified and used in functional assays described below.

FIG. 2A is an illustration of a CBB-stained SDS-PAGE gel of UnoQ-eluate containing purified NELL1 peptide. The medium was applied onto UnoQ column (Bio-Rad) as described herein. FIG. 2B shows a Western blot using anti-FLAG antibody depicting NELL1-FLAG expression in reference to a protein ladder. Peptide: 140 kDa (intracellular precursor), 130 kDa (mature form; 90 kDa peptide), 400 kDa (secreted form, homotrimer). In the example above, the productivity of the expression system was about 3 mg NELL1 peptide/L medium.

Relative to other expression systems which did not express or secrete peptide at all (such as bacterial expression, including yeast) or whose peptide production was extremely low (e.g., E. coli fused peptide system, CHO-dhfr cells, >10 mcg/L) production with the systems described (mammalian and insect cells) was surprisingly and substantially more effective at producing large amounts of functional protein.

Expression and Purification of Recombinant Rat NELL1 Protein. For production of the C-terminally FLAG-tagged NELL1 peptide by insect cells, a pIZT-NELL1-FLC plasmid was constructed by inserting the rat NELL1 cDNA fused to a FLAG epitope sequence derived from the pTB701-NELL1-FLC plasmid (Kuroda, BBRC, 265, 752-757, 1999) into insect expression vector pIZT/V5-His (Invitrogen). Furthermore, NELL1 original secretory signal sequence was replaced to honeybee mellitin signal sequence using PCR methods. High Five cells were purchased from Invitrogen, and were cultured in High Five Serum-Lree Medium (Invitrogen). High Five cells were transfected with the pIZT-NELL1-FLC plasmid using LuGene6 (Roche). Lorty-eight hours after transfection, cells were selected with 400 mg/ml of Zeocin (Invitrogen). Replace selective medium every 3 to 4 days until the stable expression cell line was established. NELL1 secretion was confirmed using immunoprecipitation and Western blot analyses. High five cells were found to express NELL1 peptides (140-kDa) in the culture medium.

The recombinant rat NELL1-FLC peptide was purified from the culture medium of Zeocin-resistant High Five cells by anion exchange chromatography using a UNO Q-1 column (Bio-Rad). NELL1 peptide was eluted at 500 mM NaCl.

For production of the C-terminally FLAG-tagged NELL1 peptide by COS7 cells, a pcDNA3.1-NELL1-FLC plasmid was constructed by inserting the rat NELL1 cDNA linked to a LLAG epitope sequence derived from the pTB701-NELL1-FLC plasmid into mammalian expression vector pcDNA3.1 (Invitrogen). COS7 cells were cultured in DMEM supplemented with 10% FBS. COS7 cells were transfected with the pcDNA3.1-NELL1-FLC using the endogenous NELL signal peptide plasmid and using electroporation method. Forty-eight hours after transfection, culture medium was subjected to immunoprecipitation and Western blot analyses for NELL1 peptide.

FIG. 2C is an illustration of a CBB-stained SDS-PAGE gel of UnoQ-eluate including NELL1-FLAG. These expression studies showed that COS cells did not express functional NELL peptide, without modifying the N terminal of the NELL to increase secretion efficiency such as including a signal sequence. FIG. 2D is an illustration of a Western blot using anti-FLAG antibody depicting NELL1-FLAG expression.

Expression and Purification of Recombinant Rat NELL2 Protein. For production of the C-terminally FLAG-tagged NELL2 peptide by insect cells, a pIZT-NELL2-FLC plasmid was constructed by inserting the rat NELL2 cDNA fused to a FLAG epitope sequence derived from the pTB701-NELL2-FLC plasmid into insect expression vector pIZT/V5-His (Invitrogen). High Five cells were purchased from Invitrogen, and were cultured in High Five Serum-Free Medium (Invitrogen). High Five cells were transfected with the pIZT-NELL2-FLC plasmid using FuGene6 (Roche). Forty-eight hours after transfection, cells were selected with 400 mg/ml of Zeocin (Invitrogen). Selective media was replaced every 3 to 4 days, until the stable expression cell line was established. NELL2 expression was confirmed in culture medium was confirmed using immunoprecipitation and Western blot analyses. High five cells were found to express NELL2 peptides (140-kDa) in the culture medium.

The recombinant rat NELL2-FLC peptide was purified from the culture medium of Zeocin-resistant High Five cells by anion exchange chromatography using a UNO Q-1 column (Bio-Rad). NELL2-FLC peptide was eluted at 500 mM NaCl.

Example 2 Expression of NELL1 in Mammalian Systems

The mammalian expression system used for production of rhNELL1 by non-viral DNA delivery in this invention may include, but not limit to these commonly used stable suspension systems listed in Table 1. The relatively detailed protocols including vector design, host cell line culture, transfection and selection of stable cell line as well as purification of rhNell-1 in HEK 293 and CHO system are described below, but are well known to those in the art.

TABLE 1 Mammalian Expression System for production of rhNell-1 Gene System Parental vector Leader sequence amplification CHO p3Xflag-CMV preprotrypsin No/optinal DXB11 Mp19-Lp human tPA DHFR/MTX HEK293 pSecTag immunoglobulin No/optinal NS/0 or Sp2/0 pdCs-Fc-X light chain of Ig and Fc DHFR/MTX fragment pEE12 N/A GS/MSX DHFR: dihydrofolate reductase; MTX: methotrexate; GS: glutamine synthetase; MSX: methionine sulphoximine.

A. CHO System #1

Vector Design: A cDNA fragment was ligated into the expression vector p3XFlag-CMV (Sigma). The resulting expression construct, pCMV-rhNELL3Xflag, includes a preprotrypsin leading sequence, cDNA fragment of the mature human NELL1 coding region and a 3Xflag sequences at c-terminal.

Host Cell Line: The CHO-K1 were adherent cell line and can be adapted to suspension culture in serum-free medium. The construct of pCMV-rhNell-1-3Xflag was transfected by either lipofectamin (Invitrogen) or calcium phosphates treatment. The stable cell lines were selected by adding G418 (400-600 ug/ml) into the cell culture medium for about two weeks. The stable transformants were further screened for single clones with high productivity of rhNELL1 by limiting dilution. The selected stable cell lines can be expended in laboratory or industrial scale bioreactors for rhNell-1 production.

Purification Procedure: rhNELL1 peptide containing media or cell lysate were purified through anti-flag antibody M2 (Sigma) affinity column at its native condition and eluted with 3Xflag peptide.

B. CHO System #2

Vector Design: FIG. 3A depicts the nucleic acid sequence of the cDNA construct and amino acid sequences of three different signal peptides that were used for the constructs.

Host Cell Line: The CHO-K1 were adherent cell line and can be adapted to suspension culture in serum-free medium. The construct of pcDNA3.1-hNELL1-c-myc/His, pIL2-hNELL1-c-myc/His, or pN2-hNELL1-c-myc/His were transfected by either lipofectamin (Invitrogen) or calcium phosphates treatment. The stable cell lines were selected by adding G418 (400-600 ug/ml) into the cell culture medium for about two weeks. The stable transformants were further screened for single clones with high productivity of rhNELL1 by limiting dilution. The selected stable cell lines can be expended in laboratory or industrial scale bioreactors for rhNELL1 production.

Purification Procedure: rhNELL1 peptide containing media or cell lysate were purified through immunoprecipitation through anti- c-myc agarose. FIG. 3B is a Western blot with anti-c-myc antibody detecting secreting NELL1 from transfections with different constructs after immunoprecipitation using anti-c-myc agarose. FIG. 3C is a Western blot with anti-c-myc or mouse anti-human NELL1 antibodies detecting secreting NELL1 after immunoprecipitation using rabbit anti-human Nell-1 antibody-NHS activated sepharose.

C. CHO System #3

Vector Design: Proprietary cDNA constructs expressing a NELL1 peptide was constructed according to the general procedures described above.

Host Cell Line: The proprietary CHO cell lines were adherent cell line and can be adapted to suspension culture in serum-free medium. The proprietary constructs were transfected. The stable cell lines were selected by adding appropriate factors into the cell culture medium for about two weeks. The stable transformants were further screened for single clones with high productivity of rhNELL1 by limiting dilution. The selected stable cell lines can be expended in laboratory or industrial scale bioreactors for rhNELL1 production.

Purification Procedure: rhNELL1 peptide containing media or cell lysate were purified through analytical and preparative protein purifications methods well known to those in the art (e.g., size, exclusion chromatography, ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, high performance liquid chromatography,

Concentration Procedure: rhNELL1 was concentrated using lyophilization or ultrafiltration.

D: HEK293 System

Vector Design: A cDNA fragment was ligated into the expression vector pSecTagA (Invitrogen). The resulting expression construct, pSec-hNell-1-Tag, includes a murine immunoglobulin □-chain leader sequence, cDNA fragment of the mature human NELL1 coding region and dual tag of Myc and His sequences at c-terminal.

Host Cell Line: The human embryo kidney cell line, HEK-293 which was adapted to serum-free medium and grown in suspension format, was transfected with the NELL1 peptide expression vector, pSec-hNell-1-Tag. Cells were either cultured for a couple of days as transient transfection before collecting conditioned medium for purification of rhNell-1 or treated with Zeocin (250 ug/ml) for selection of stable expression cell line. The stable transformants were further screened for single clones with high productivity of rhNell-1 by limiting dilution. The selected stable cell lines can be expended in laboratory or industrial scale bioreactors for rhNell-1 production.

Purification Procedure: rhNell-1 peptide containing media were purified through Ni²⁺ affinity column at its native condition and eluted withlM imidazole. The rhNell-1 was tested for its integrity, purity and bioactivity after extensively dialysis against at least 1000 volumes of PBS (pH 7.4) at 4° C. for 20 hrs.

In addition, the modifications of parental vectors for replacing existing leader sequence with a new one such as rat serum albumin, CD33, tPA and human interlukin-2 leader sequence or adding gene amplification target such as DHFR or GS into the backbone sequence will result in new expression vectors and systems. In this invention, the native signal peptide of human Nell-1 is not effective enough to guide the protein secretion and sometimes even the external leading sequence didn't work well, either. Thus, the construction of expression vector with in frame fusion of a small natural secretory protein such as human granulocyte-macrophage colony stimulating factor (GM-CSF) by a spacer containing intraprotein His tag and proteolytic cleavage site as “MPHHHHHHGGGDDDDKDPM” might be needed. The epitope tags used for purification of NELL1 can be one of the following: 6XHistidines, 3XFlag, Myc, GST (glutathione S-transferase), EGFP or CTHS (C-terminal half of SUMO which stands for small ubiquitin modifying protein) etc, but also could be dual of His plus Myc as listed plasmid pSecTag in Table 1 (supra).

Furthermore, the dicistronic or multicistronic vectors using IRES might be constructed for regulatory or inducible expression of rhNell-1 under certain circumstances. The genetic modifications of host cell lines for gaining longer lasting proliferation and delayed apoptosis or compatible with special requests such as Tet (tetracycline) inducible system and Flp-In specific site integration system will be considered for improvement of rhNell-1 production.

Besides the stable expression of system for production of rhNell-1 mentioned above, we would not exclude the possibility to establish a large-scale transient transfection (LST) approach using multi-milligram purified plasmid vector (pREP4) to transfect HEK 293 or BHK suspension cells with cationic polymer PEI as backup alternative or complimentary to stable system.

Example 3 Purification of NELL2 Protein from Culture Medium

High Five cells carrying pIZT-FLC-NELL2 were cultured for about three days in serum free culture medium (1 L). The culture medium was centrifuged at. 3000×g for 5 minutes and the supernatant was collected. PMSF was added to a final concentration of 1 mM. Saturated ammonium sulfate solution (80% saturation (v/v) was added and the solution kept at 4 degrees for 1 hour. The solution was centrifuged at 15000×g for 30 min. and precipitate collected. Precipitate was dissolved in 50 ml of 20 mM Tris-HCl (pH 8.0), 1 mm EDTA at 4 degree and applied onto an anion-exchange chromatography UnoQ column (6 ml, Bio-Rad) equilibrated in 20 mM Tris-HCl (pH 8.0), 1 mM EDTA at 4 degree (1 ml/min speed by FPLC (Amersham-Pharmacia). The column was thoroughly washed with the same buffer.

The binding protein was then eluted by the gradation from 0 M to 1.5 M NaCl in the same buffer. The NELL2-FLAG fractions were identified by Western blotting using anti-Flag M2 (Sigma) Ab. The positive fractions were collected into one tube. Final product was dialyzed in the seamless cellulose tube (Wako, cutoff MW 12000) against 1 L PBS for overnight at 4 degree. The product was stored at −70 degree.

Example 4 Cell Line Development and Expression of NELL-1

A series of Chinese Hamster Ovary (CHO) cell lines expressing recombinant human Nell-1 protein were developed according to procedures described above or procedures known in the art. The cell lines were screened according to known methods (please see references 1-8, below). The top 12 clones were cultured in a medium to amplify expression. The cell pools exhibiting the highest expression of human Nell-1 by ELISA were subcloned. They were then subjected to amplification, after which the cell pools were rescreened by ELISA. From this procedure, the ten highest expressing MTX-amplified subclones from transfection were expanded.

The six highest expressing subclones were adapted to grow in protein-free medium. All serum-free adapted clones were evaluated for Nell-1 expression by ELISA and by SDS gel. The clones that were observed to express the best at 1,000 nM MTX were identified as N2-1.9E10.1F9.4C2, N2-1.9E10.1F9.5H6, N2-1.9E10.1F9.5H2. All expressed Nell-1 protein at levels=40 ug/ml by ELISA.

-   (1) Urlaub, G. & Chasin, L. (1980) Isolation of Chinese Hamster Cell     Mutants Deficient in Dihydro folate Reductase Activity. Proc Natl.     Acad. Sci. 77:4216-4220 -   (2) Kao, F.-T. and Puck, T. T. (1967) Genetics of somatic mammalian     cells. IV. Properties of Chinese hamster cells with respect to the     requirement for proline. Genetics 55:513-524 -   (3) Wigler, M., Perucho, M., Kurtz, D., Dana, S., Pellicer, A, Axel,     R., and Silverstein, S. (1980) Transformation of mammalian genes     with an amplifiable dominant-acting gene. Proc. Natl. Acad. Sci. USA     Vol 77: 3567-3570. -   (4) Bourouis, M. and Jarry, B. (1983) Vectors containing a     prokaryotic dihydrofolate reductase gene transform Drosophila cells     to methotrexate resistance. EMBO J. 2:1099-1104. -   (5) Ringold, G., Dieckmann, B., and Lee, F. (1982) Co-expression and     amplification of dihydrofolate reductase cDNA and the Escherichia     coli XGPRT gene in Chinese hamster ovary cells. Journal of Molecular     and Applied Genetics, Vol. 1:3: 165-175. -   (6) Gasser C. S., Simonsen C. C., Schilling J. W.,     Schimke R. T. (1982) Expression of abbreviated mouse dihydrofolate     reductase genes in cultured hamster cells. Proc. Natl. Acad. Sci. V     79: 6522-6526. -   (7) Kaufman, R. J., Wasley, L. C., Spiliotes, A. J., Gossels, S. D.,     Latt, S. A., Larsen, G. R. and Kay, R. M. (1985) Co-amplification     and co-expression of human tissue type plasminogen activator and     murine dihydrofolate reductase sequences in Chinese hamster ovary     cells. Mol. Cell. Biol. Vol 5: 1750-1759. -   (8) Simonsen, C. C. and Levinson, A. D. (1983) Isolation and     expression of an altered mouse dihydrofolate reductase cDNA. Proc.     Natl. Acad. Sci. U.S.A. 80: 2495-2499.

While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that changes and modifications can be made without departing from this invention in its broader aspects. Therefore, the claims are to encompass within their scope all such changes and modifications as fall within the true sprit and scope of this invention. 

What is claimed is:
 1. A method of purifying a NEL-like protein (NELL) peptide comprising: (a) providing a crude solution comprising the NELL peptide, and (b) subjecting the crude solution to a chromatography process to obtain a purified NELL peptide, wherein the chromatography process uses a medium comprising a metal ion bearing two or more charges.
 2. The method of claim 1, wherein the chromatography process comprises contacting the crude solution to a chromatography membrane or column.
 3. The method of claim 1, wherein the method further comprises, prior to step (a): (i) transfecting a mammalian cell with a nucleic acid construct comprising a nucleic acid encoding the NELL peptide in frame with a nucleic acid encoding a heterologous, non-insect secretory signal peptide; (ii) culturing the mammalian cell under conditions that permit expression of the NELL peptide; and (iv) collecting media comprising the NELL peptide secreted from the cell to form the crude solution.
 4. The method of claim 1, further comprising testing bone formation-inducing activity of the purified NELL peptide.
 5. The method of claim 1, wherein the chromatography process comprises membrane or affinity chromatography.
 6. The method of claim 1, wherein the metal ion is calcium ion.
 7. The method of claim 3, wherein the non-insect secretory signal peptide is a plant secretory signal peptide or an animal secretory signal peptide.
 8. The method of claim 7, wherein the animal secretory signal peptide is a mammalian secretory signal peptide.
 9. The method of claim 8, wherein the mammalian secretory signal peptide is a human secretory signal peptide.
 10. The method of claim 3, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.
 11. The method of claim 3, wherein the nucleic acid encoding the NELL peptide encodes NELL1 or NELL2.
 12. The method of claim 11, wherein the nucleic acid encoding NELL1 comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.
 13. The method of claim 11, wherein the nucleic acid encoding NELL2 comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. 